Compositions and methods for detecting stress-inducible proteins

ABSTRACT

Compositions and methods for detection of the stress-inducible Hsp70B′ protein are disclosed. These include antibodies directed against particular amino acid regions of Hsp70B′ and various peptides corresponding, or antigenically equivalent, to the regions. The ability to generate anti-Hsp70B′ antibodies to defined epitopes permits a variety of in vitro and in vivo uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional and claims the benefit ofpriority under 35 U.S.C. § 120 of U.S. Ser. No. 11/089,878, filed Mar.25, 2005, now U.S. Pat. No. 7,138,247 which is a divisional of U.S. Ser.No. 09/733,179, filed Dec. 7, 2000, now U.S. Pat. No. 6,964,851, whichclaims the benefit of U.S. Ser. No. 60/169,535, filed Dec. 7, 1999, allof which are herein incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention features compositions and methods for detectingthe stress-inducible protein Hsp70B′. More specifically, the inventionfeatures antibodies that specifically bind Hsp70B′ or fragments,antigenically equivalent portions, or epitopes thereof.

BACKGROUND OF THE INVENTION

Cells within most organisms have evolved a mechanism known as the“cellular stress response” to cope with adverse changes in theirenvironment. The response is a universal cellular defense mechanism thatresults in increased expression of a class of proteins referred to as“heat shock” or “stress” proteins. The conditions that trigger theresponse include: a rise in temperature, hypoxia, irradiation,nutritional deficiencies, acute exercise, infection, or exposure to ametabolic insult such as a proinflammatory cytokine, a heavy metal, anamino acid analogue, or a metabolic poison (Kelly et al., J. Appl.Physiol. 81:2379-2385, 1996; Minowada and Welch, J. Clin. Invest.95:3-12, 1995).

Stress proteins are also essential for normal cellular function and manyare constitutively expressed. They are believed to help regulate thecell cycle and cellular differentiation and to maintain the cell atcritical stages of organ development (Birnbaum, Springer Semin.Immunopathol. 17:107-118, 1995). Some stress proteins are molecularchaperones that facilitate the correct folding or conformation ofnascent polypeptides, direct intracellular trafficking of proteins,protect proteins against denaturation, and assist in the renaturation ofunfolded proteins (Macario, Int. J. Clin. Lab Res. 25:59-70, 1995).Stress proteins also participate in antigen presentation and nuclearreceptor binding and act as anti-apoptotic agents.

The HSP70 family of stress proteins includes at least 11 different genesthat encode highly related protein isoforms ranging in size from 66 kDato 110 kDa (Tavaria et al., Cell Stress & Chaperones 1:23-28, 1996).Members of this family help regulate protein synthesis andtranslocation, protein-protein interactions, thermotolerance, andprotein degradation (Mangurten et al., Cell Stress & Chaperones2:168-174, 1997).

Members of the human hsp70 gene family also display considerablestructural and sequence similarity; the greatest sequence divergence isin the untranslated regions and extreme C-terminal coding regions (Leunget al., Genomics 12:74-79, 1992). Individual Hsp70 family members differin their levels of basal expression and are induced under differentconditions (Leung et al., Genomics 12:74-79, 1992). The majority ofHsp70 protein isoforms are synthesized constitutively, but theirexpression may be up-regulated following exposure to an environmentalinsult. These proteins bind ATP through an ATP-binding cassette at theirN-terminus and have a large C-terminus peptide-binding domain (Maio etal., Guidebook to Molecular Chaperones and Protein-Folding Catalysts,Sambrook & Tooze Publication, Oxford University Press, 1997). Thispeptide binding function allows Hsp70proteins to play a significant rolein the protection and folding of nascent proteins after synthesis, inthe translocation of proteins through membranes, and in the protectionand repair of stress-induced protein damage (Minowada and Welch, J.Clin. Invest. 95:3-12, 1995).

Members of the human HSP70 protein family associate with distinctcellular compartments. Prominent family members include: i) theconstitutive Hsc70 (or cognate) protein, which is present within thecytosol and nucleus, ii) the highly stress-inducible Hsp70A protein,which is present within the cytosol, nucleus, and nucleolus (thisprotein is present at basal levels in unstressed human cells), iii) thestrictly stress-inducible Hsp70B′ protein and its closely relatedisoform Hsp70B, iv) the constitutive glucose regulated 78 kDa protein(or BiP), which is present within the lumen of the endoplasmicreticulum, and v) the glucose regulated 75 kDa protein (Grp75 or mtHsp75), which is present within mitochondria (Tavaria et al., Cell Stress &Chaperones 1:23-28, 1996).

Antibodies have been raised against Hsp70 family members that areexpressed at basal levels and whose expression can be induced to highlevels (i.e., Grp75, and Hsp70 A) and to the constitutive Hsp70 familymembers (e.g., Hsc70 , BiP). However, there are no antibodies thatspecifically bind the strictly inducible Hsp70 B′ protein or itshomologue, Hsp70B. Thus, immunological based assays (such asimmunoblotting, EIA, and immunohistochemistry)) have been practiced withantibodies that are not strictly stress inducible. The results obtainedwith these assays are ambiguous because the Hsp70 family of proteins isso complex. While there is some indication that Hsp70A and Grp70 areupregulated under conditions of stress, the significant basal level ofthe inducible Hsp70A protein in normal tissue, neoplastic tissue, andcell lines (Bachelet et al., Cell Stress & Chaperones 3:168-176, 1998;Bratton et al., Int. J. Hyperthermia 13:157-168, 1997; Sztankay et al.,Journal of Autoimmunity 7:219-230, 1994), and an extreme variation inbaseline levels in unstressed cells (Pockley et al., Immunol. Invest.27:367-77, 1998), confounds interpretation limits the utility ofprevious studies. The dual function of the Hsp70 family is alsoproblematic. Hsp70 stress proteins function both constitutively (byperforming cellular “housekeeping” functions) and inductively (byresponding to adverse changes to the cellular environment). The assaysdeveloped to date assess incremental increases in an already expressedprotein (Hsp70A) but, because preexisting basal levels fluctuate somuch, the results are difficult to interpret. Thus, there is a need forantibodies that specifically bind the strictly stress inducible Hsp70B′protein. The novel compositions of the present invention fulfill thisneed.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the identification ofimmunogenic peptide sequences from the human Hsp70B′ protein. Antibodiesthat specifically bind this protein can be used to distinguish betweenthe expression of HSC70/HSP70 proteins, which occurs while a cell isfunctioning normally and when it is responding to stress, and theHsp70B′ protein, which is only produced in response to stress (i.e.,substances or events that are detrimental to the health of the cell ororganism). Hsp70B′ is unique among Hsp70 family proteins because neitherhsp70B′ mRNA nor Hsp70B′ protein has been detected in unstressed cells.Accordingly, the present invention features compositions and methods fordetermining whether a cell (or a population of cells, such as those incell culture or within a tissue) expresses Hsp70B′. A positive reactionto an Hsp70B′ antibody not only provides evidence of stress in aparticular cell, but also provides an indication of the general state ofthe health of the organism in which that cell resides (or from which itwas obtained). As described below, the compositions of the invention(e.g. antibodies that specifically bind an Hsp70B′ protein, an antigenicfragment, or an epitope thereof) can be used to determine whether a cell(e.g. a human cell), an organ (e.g. the skin or the liver), or an entireorganism (e.g. a human) has been exposed to a stressor.

The invention also features peptides that correspond to, or areantigenically equivalent to, regions of the Hsp70B′ protein. The peptidecan consist of five or more (e.g., 5, 6, 7, 8, 10, or 12) consecutiveamino acids from Hsp70B′ protein (beginning at the second, fourth,sixth, or eighth residue), such as the following amino acid sequences:(1) VPGGSSCGTQARQGDPSTGPI (SEQ ID NO:1) (e.g., CGTQARQGDPSTGPI (SEQ IDNO:2) and CGTQARQGDPST (SEQ ID NO:3)); (2) RDKIPEEDRRKMQDKC (SEQ IDNO:4) (e.g., RDKIPEEDRRKMQ (SEQ ID NO:5); when these peptides are linkedto keyhole limpet hemocyanin (KLH), they can include cysteine residues);(3) AHVFHVKGSLQEESLRDKIPEEDRRKMQ (SEQ ID NO:6) (e.g., AHVFHVKGSLQEES(SEQ ID NO:7); (4) MQAPRELAVGID (SEQ ID NO:8), which is located in theN-terminal of Hsp70B′ and, when linked to KLH includes a cysteineresidue (i.e., MQAPRELAVGID(C) (SEQ ID NO:9)); (5) GSLQEESLRDKIPEE (SEQID NO:10).

The peptides of the invention can contain at least one amino acidsubstitution (e.g., 1, 2, or 3 of the residues in the peptides of theinvention can be replaced with another amino acid residue;alternatively, up to about 50% (e.g., 10%, 25%, 30%, 40% or 50%) of theresidues in the peptides can be substituted). The substitution canconstitute a conservative amino acid substitution. Conservativesubstitutions include interchanges of alanine and valine, valine andisoleucine, leucine and isoleucine, aspartic acid and glutamic acid,threonine and serine, and others of a similar nature (for example, anyin which the neutral, positive or negative charge of the original aminoacid residue is maintained). Conservative amino acid substitutions arewell known to those of ordinary skill in the art. Preferably, peptidescontaining substitutions will be antigenically equivalent to thenaturally occurring peptide sequence (i.e. a peptide containing asubstitution will have a relative titre index that is no less than halfas great as the relative titre index of the naturally occurringpeptide). The peptides of the invention can also be attached to acarrier (e.g., KLH or ovalbumin) that enhances their immunogenicity orcirculating half-life. Unless otherwise noted, a “protein” is afull-length protein (e.g., a full length Hsp70B′ protein) and a“peptide” is a portion of a full-length protein (e.g. five or moreconsecutive amino acid residues present within the Hsp70B′ protein). A“polypeptide” may be either a protein or peptide.

In related aspects, the invention features antibodies that specificallybind Hsp70B′ or one of the Hsp70B′ peptides disclosed herein and methodsof obtaining those antibodies. The antibodies can be polyclonal ormonoclonal antibodies, and can be produced by methods well known tothose of ordinary skill in the art. These methods typically includeimmunizing an animal with Hsp70B′ or an Hsp70B′ peptide, but can becarried out instead by immunizing an animal with a nucleic acid moleculethat encodes Hsp70B′ or an Hsp70B′ peptide.

The antibodies of the invention may be used to specifically bind, andthereby detect, Hsp70B′ in virtually any immunoassay (e.g., an assaycarried out by binding Hsp70B′ proteins or peptides that are immobilized(e.g. on a membrane or column) or present in a cell (by, e.g.,immunohistochemistry)). Accordingly, the invention features kits thatinclude antibodies that specifically bind an Hsp70B′ protein or peptide.The kits can also optionally include an Hsp70B′ protein or peptide (as apositive control), an irrelevant protein (i.e., one to which thesupplied antibody does not bind; as a negative control), secondaryantibodies, other reagents, buffers, or solutions, and instructions.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are plots representing the displacement curves for Hsp70B′antibodies in the presence of Hsp70B′ standard and HSP70 homologues. (A)is the CB2 displacement curve, (B) is the CD displacement curve, and (C)is the 70B′ WP displacement curve.

FIG. 2 is a representation of the human Hsp70B′ amino acid sequence (SEQID NO:11).

DETAILED DESCRIPTION

The invention features immunogenic peptides whose sequence is present inthe Hsp70B′ protein or whose sequence varies from the sequence of theHsp70B′ protein in such a limited way as to remain an antigenicequivalent of the naturally occurring peptide. For example, an Hsp70B′protein or peptide that contains one or more amino acid substitutions(e.g. one or more conservative amino acid substitutions) can beantigenically equivalent to the naturally occurring Hsp70B′ protein orpeptide fragments thereof. Proteins and peptides that, uponadministration to an animal, elicit the production of antibodies thatspecifically bind to Hsp70B′ protein include the following: (1)VPGGSSCGTQARQGDPSTGPI (SEQ ID NO:1) (e.g., CGTQARQGDPSTGPI (SEQ ID NO:2)and CGTQARQGDPST (SEQ ID NO:3)); (2) RDKIPEEDRRKMQDKC (SEQ ID NO:4)(e.g., RDKIPEEDRRKMQ (SEQ ID NO:5); when these peptides are linked tokeyhole limpet hemocyanin (KLH), they can include N-terminal cysteineresidues); (3) AHVFHVKGSLQEESLRDKIPEEDRRKMQ (SEQ ID NO:6) (e.g.,AHVFHVKGSLQEES (SEQ ID NO:7); (4) MQAPRELAVGID (SEQ ID NO:8), which islocated in the N-terminal of Hsp70B′ and, when linked to KLH includes aC-terminal cysteine residue (i.e., MQAPRELAVGID(C) (SEQ ID NO:9)); (5)GSLQEESLRDKIPEE (SEQ ID NO:10); and the Hsp70B′ protein (SEQ ID NO:11).

Portions of any of these peptides can also be used to generateHsp70B′-specific antibodies. More specifically, five or more consecutiveamino acid residues (i.e., amino acid residues linked to one another bypeptide bonds in the same sequential order as they appear in thenaturally occurring sequence) can be used. The starting point can beanywhere within the sequence of Hsp70B′ or the Hsp70B′ peptidesdisclosed herein, up to the fifth-to-last amino acid residue. Peptidesbased on the sequences disclosed herein can contain at least 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 18, 20, 25, or 28 consecutive amino acidresidues. Moreover, they can begin at, for example, the second, fifth,ninth, tenth, or twelfth residue in any of the peptides disclosedherein.

The Hsp70B′ protein or any of the Hsp70B′ peptides can be attached to acarrier that enhances their immunogenicity. The carrier is any substancethat, when attached to the protein or peptide, results in the productionof more antibodies than when it is omitted from the protein or peptide.The carrier can be attached to the protein or peptide covalently ornoncovalently so long as the two entities remain attached to one anotherwhen administered to an animal. More specifically, the carrier can be anamino acid-based substance such as keyhole limpet hemocyanin (KLH).Regardless of the means of attachment, one or more groups (e.g.,chemically reactive groups or one or more amino acid residues) can beused to facilitate bonding between the protein or peptide and thecarrier. For example, a cysteine residue can be added to either end ofHsp70B′ or to any of the Hsp70B′ peptides described herein to facilitatecoupling with a carrier. If desired, more than one carrier can be used,and a spacer (e.g. one or more amino acid residues) can be added betweenthe protein or peptide and the carrier.

Antibodies may generally be prepared by any of a variety of techniquesknown to those of ordinary skill in the art (see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).For example, an immunogen that includes a Hsp70B′ peptide is initiallyinjected into suitable animals (e.g., mice, rats, rabbits, sheep andgoats) according to a predetermined schedule with one or more boosterimmunizations, and blood samples are obtained from the animalsperiodically. Polyclonal antibodies specific for the Hsp70B′ peptide canthen be purified from the antisera by, for example, affinitychromatography in which the same peptide sequence administered iscoupled to a suitable solid support.

To obtain Hsp70B′-specific antibodies, an animal can be immunized withHsp70B′ or one of the Hsp70B′ peptides disclosed herein. The term“immunized” refers to at least a first administration of antigen, andoptionally includes subsequent administration (e.g., a second or thirdadministration) and additional periodic boosting. Immunization typicallyincludes administration of the antigen (i.e. an Hsp70B′ protein or afragment or epitope thereof that evokes an immune response) and anadjuvant. The Hsp70B′ protein or peptide administered can be purifiedfrom a natural source, chemically synthesized, or recombinantlyproduced. Regardless of the length of the amino acid sequence used toimmunize an animal, sera (or antigen-specific B cells or other antibodycontaining fluids) are collected, and the antibody response isevaluated, typically by immunoassay. High-titre preparations aregenerally pooled and the protein-specific or peptide-specific antibodyis purified on an immunoaffinity column to which the protein or peptideis immobilized. Where full length Hsp70B′ protein (or any portion of theprotein that is longer than the peptides disclosed herein) is used togenerate antibodies by immunization or otherwise, an Hsp70B′ peptide(such as those disclosed herein) is typically used to purify theantibodies. The discovery of amino acid sequences within Hsp70B′ thatcan yield antibodies that distinguish Hsp70B′ from other Hsp70 familymembers permits antibodies to be obtained in many ways known to those ofordinary skill in the art.

In addition to polyclonal antibodies, the antibodies generated can bemonoclonal antibodies, fragments of polyclonal or monoclonal antibodiessuch as F(ab′)₂, and Fab fragments, as well as any naturally occurringor recombinantly produced binding partners (i.e., molecules thatspecifically bind Hsp70B′). In addition to the Hsp70B′ protein orpeptide (which functions as an antigen), the composition administeredcan include a carrier vehicle and immunostimulatory substances thatenhance immunogenicity (e.g., adjuvants). The carrier vehicle caninclude aluminum salts, water-in-oil emulsions, biodegradable oilvehicles, oil-in-water emulsions, biodegradable microcapsules, andliposomes. The immunostimulatory substances can include includeN-acetylmuramyl-L-alanine-D-isoglutamine (MDP), lipopoly-saccharides(LPS), glucan, IL-12, GM-CSF, gamma interferon, and IL-15.

Monoclonal antibodies specific for Hsp70B′ peptides can be prepared, forexample, using the technique of Kohler and Milstein (Eur. J. Immunol.6:511-519, 1976), and improvements thereto. Briefly, these methodsinvolve the preparation of immortal cell lines that produce antibodieshaving the desired specificity (i.e., reactivity with the Hsp70B′peptide of interest). Such cell lines may be produced, for example, fromspleen cells obtained from an immunized animal. The spleen cells arethen immortalized by, for example, fusion with a myeloma cell fusionpartner, preferably one that is syngeneic with the immunized animal. Forexample, the spleen cells and myeloma cells may be combined with anagent that promotes membrane fusion (e.g., polyethylene glycol or anonionic detergent), and then plated at low density on a selectivemedium that supports the growth of hybrid cells, but not myeloma cells.The selection technique can be HAT (hypoxanthine, aminopterin,thymidine) selection. After a sufficient time (typically 1 to 2 weeks),colonies of hybrids are observed. Single colonies are selected andtested for binding activity against the polypeptide. Hybridomas havinghigh reactivity and specificity are preferred. Monoclonal antibodies maybe isolated from the supernatants of growing hybridoma colonies.Accordingly, such hybridomas and the monoclonal antibodies they produce(i.e., monoclonal antibodies that specifically bind to Hsp70B′ orHsp70B′ peptides) are specifically encompassed by the present invention.

Techniques that enhance the yield of antibodies are known in the art andcan be used in the context of the present invention. For example, thehybridoma cell line can be injected into the peritoneal cavity of asuitable vertebrate host, such as a mouse, and monoclonal antibodies maythen be harvested from the ascites fluid or the blood of that host.Contaminants can be removed by conventional techniques, such aschromatography, gel filtration, precipitation, and extraction. Forexample, anti-Hsp70B′ antibodies can be 10 purified by chromatography onimmobilized Protein G or Protein A using standard techniques.

Instead of administering Hsp70B′ or Hsp70B′ peptides, animals can beindirectly immunized by administering nucleic acid molecules encodingHsp70B′ or a Hsp70B′ peptide. Accordingly, nucleic acid molecules thatencode the Hsp70B′ peptides disclosed herein, alone or in the context ofan expression, and cells that contain those molecules are within thescope of the invention. These nucleic acid molecules can be deliveredwith recombinant viral vectors (e.g., retroviruses (see WO 90/07936, WO91/02805, WO 93/25234, WO 93/25698, and WO 94/03622), adenovirus (seeBerkner, Biotechniques 6:616-627, 1988; Li et al., Hum. Gene Ther.4:403-409, 1993; Vincent et al., Nat. Genet. 5:130-134, 1993; and Kollset al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994), pox virus (seeU.S. Pat. Nos. 4,769,330 and 5,017,487; and WO 89/01973)), naked DNA(see WO 90/11092), nucleic acid molecule complexed to a polycationicmolecule (see WO 93/03709), and nucleic acid associated with liposomes(see Wang et al., Proc. Natl. Acad. Sci. USA 84:7851, 1987). The DNA canbe linked to killed or inactivated adenovirus (see Curiel et al., Hum.Gene Ther. 3:147-154, 1992; Cotton et al., Proc. Natl. Acad. Sci. USA89:6094, 1992). Other suitable compositions include DNA-ligand (see Wuet al., J. Biol. Chem. 264:16985-16987, 1989) and lipid-DNA combinations(see Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989). Inaddition, the efficiency with which naked DNA is taken up by cells canbe increased by coating the DNA onto biodegradable beads.

In some cases, antigen-binding fragments of antibodies are preferred.These fragments include Fab fragments, which may be prepared usingstandard techniques (e.g., by digestion with papain to yield Fab and Fcfragments). The Fab and Fc fragments can be separated by affinitychromatography (e.g., on immobilized protein A columns), using standardtechniques. See, e.g., Weir, D. M., Handbook of Experimental Immunology,1986, Blackwell Scientific, Boston.

Multifunctional fusion proteins having specific binding affinities forpre-selected antigens by virtue of immunoglobulin V-region domainsencoded by DNA sequences linked in-frame to sequences encoding variouseffector proteins are known in the art, for example, as disclosed inEP-B1-0318554 and U.S. Pat. Nos. 5,132,405, 5,091,513, and 5,476,786.Such effector proteins include polypeptide domains that can be used todetect binding of the fusion protein by any of a variety of routinelypracticed techniques, including but not limited to a biotin mimeticsequence (see, e.g., Luo et al., J. Biotechnol. 65:225, 1998 andreferences cited therein), direct covalent modification with adetectable labeling moiety, non-covalent binding to a specific labeledreporter molecule, enzymatic modification of a detectable substrate orimmobilization (covalent or non-covalent) on a solid-phase support.

Single chain antibodies that can be used in the methods described hereincan also be generated and selected by a method such as phage display(see, e.g., U.S. Pat. No. 5,223,409, Schlebusch et al., Hybridoma16:47,1997, and references cited therein). Briefly, in this method, DNAsequences are inserted into the gene III or gene VIII gene of afilamentous phage, such as M13. Several vectors with multicloning siteshave been developed for insertion (McLafferty et al., Gene 128:29-36,1993; Scott and Smith, Science 249:386-390, 1990; Smith and Scott,Methods Enzymol. 217:228-257, 1993). The inserted DNA sequences can berandomly generated or can be variants of a known binding domain forbinding to Hsp70B′ peptides. Single chain antibodies can be readilygenerated using this method. Generally, the inserts encode from 5 to 20amino acid residues. The peptide encoded by the inserted sequence isdisplayed on the surface of the bacteriophage. Bacteriophage expressinga binding domain for a Hsp70B′ peptide are selected by binding to animmobilized Hsp70B′ peptide, for example a recombinant polypeptideprepared using methods well known in the art and nucleic acid codingsequences as disclosed by Chang et al. (Proc. Nat. Acad. Sci. USA93:136, 1996) or by Kojima et al. (J. Biol. Chem. 270:21984, 1995).Unbound phage is removed by a wash, typically containing 10 mM Tris, 1mM EDTA, and without salt or with a low salt concentration. Bound phageis eluted with a salt containing buffer, for example. The NaClconcentration is increased in a step-wise fashion until all the phage iseluted. Typically, phage binding with higher affinity will be releasedby higher salt concentrations. Eluted phage is propagated in thebacteria host. Further rounds of selection may be performed to selectfor a few phage binding with high affinity. The DNA sequence of theinsert in the binding phage is then determined. Once the predicted aminoacid sequence of the binding peptide is known, sufficient peptide foruse as an antibody specific for a human Hsp70B′ protein or peptide canbe made either by recombinant means or synthetically. Recombinant meansare used when the antibody is produced as a fusion protein. The peptidecan also be generated as a tandem array of two or more similar ordissimilar peptides, in order to maximize affinity or binding.

Antibodies that specifically bind an Hsp70B′ protein (e.g., a murine,porcine, bovine, equine, or human Hsp70B′ protein) can be used in vitroor in vivo to evaluate, diagnose, or form a prognosis regarding aspecific cell or a disease state. These antibodies are molecular markersof exposure to a stressful environment (e.g. an environment where thetemperature is increased beyond physiological norms, there is a shortageof oxygen, or an infectious organism) or substance (e.g. a toxin, aproinflammatory cytokine, a heavy metal, an amino acid analogue, or ametabolic poison). Notably, the antibodies can serve as markers of anadverse sub-lethal effect of stress. To detect an antigenic determinantreactive with an antibody specific for a human Hsp70B′ peptide, thedetection reagent is typically an antibody, which may be prepared asdescribed herein. The variety of assay formats known to those ofordinary skill in the art include, but are not limited to, enzymeimmunoassay (EIA), enzyme linked immunosorbent assay (ELISA),radioimmunoassay (RIA), immunofluorimetry, immunoprecipitation,equilibrium dialysis, immunodiffusion and other techniques. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; Weir, D. M., Handbook of Experimental Immunology,1986, Blackwell Scientific, Boston. For example, the assay may beperformed in a Western blot format in which a protein preparation fromthe biological sample is subjected to gel electrophoresis, transferredto a suitable membrane, and allowed to react with the antibody. Thepresence of the antibody can then be detected using a suitable detectionreagent, as is well known in the art and described below.

The assay can also involve antibodies immobilized on a solid support(e.g., a test well in a microtiter plate, a nitrocellulose filter orother suitable membrane, a bead or disc, such as glass, fiberglass,latex or a plastic such as polystyrene or polyvinylchloride). Theimmobilized antibody can bind to the target Hsp70B′ peptide or proteinand thereby separate it from substantially all of the rest of thesample. The bound polypeptide can then be detected with a secondantibody reactive with a distinct polypeptide antigenic determinant, forexample, a reagent that contains a detectable reporter moiety. Forexample, the immobilized antibody and the secondary antibody which eachrecognize distinct antigenic determinants may be two monoclonalantibodies. Alternatively, a competitive assay may be utilized, in whicha polypeptide is labeled with a detectable reporter moiety and allowedto bind to the immobilized polypeptide specific antibody afterincubation of the immobilized antibody with the sample. The extent towhich components of the sample inhibit the binding of the labeledpolypeptide to the antibody is indicative of the reactivity of thesample with the immobilized antibody, and as a result, indicative of thelevel of Hsp70B′ polypeptide in the sample.

In some cases, the assay used to detect Hsp70B′ polypeptides in a sampleis a two-antibody sandwich assay. This assay can be performed by firstcontacting a Hsp70B′ antibody that has been immobilized on a solidsupport (commonly the well of a microtiter plate) with the biologicalsample. Soluble molecules that naturally occur in the sample and have anantigenic determinant that is reactive with the antibody will bind tothe immobilized antibody and thereby form an antigen-antibody complex oran immune complex. A 30-minute incubation at room temperature isgenerally sufficient for complex formation. Unbound constituents of thesample are then removed from the immobilized immune complexes and asecond antibody specific for a Hsp70B′ polypeptide is added. Theantigen-combining site of the second antibody does not competitivelyinhibit binding of the antigen-combining site of the immobilized firstantibody. The second antibody may be detectably labeled as providedherein, such that it may be directly detected. Alternatively, the secondantibody may be indirectly detected with a labeled secondary (or “secondstage”) anti-antibody, or by using a specific detection reagent asprovided herein. Notably, the methods of the invention need not belimited to any particular detection procedure. Those familiar withimmunoassays understand that there are numerous reagents andconfigurations for immunologically detecting a particular antigen in atwo-antibody sandwich immunoassay.

When a two-antibody sandwich assay is used, the first, immobilizedantibody specific for a Hsp70B′ polypeptide and the second antibodyspecific for a Hsp70B′ polypeptide can both be polyclonal antibodies.Alternatively, the first, immobilized antibody specific for Hsp70B′polypeptide can be a monoclonal antibody and the second antibodyspecific for a Hsp70B′ polypeptide can be a polyclonal antibody andvice-versa. It can be preferable, however, to carry out the assay withthe first, immobilized antibody and the second anti-Hsp70B′ antibodybeing monoclonal antibodies. In yet other configurations, the first,immobilized antibody and/or the second antibody may be any of the kindsof antibodies known in the art (for example, Fab fragments, F(ab′)₂fragments, immunoglobulin V-region fusion proteins or single chainantibodies). Those of ordinary skill in the art will appreciate that thepresent invention can be practiced with other antibody forms, fragments,derivatives, and the like.

The second antibody may contain a detectable reporter moiety or labelsuch as an enzyme, dye, radionuclide, luminescent group, fluorescentgroup or biotin, or the like. The amount of the second antibody thatremains bound to the solid support is then determined using a methodappropriate for the specific detectable reporter moiety or label. Forradioactive groups, scintillation counting or autoradiographic methodsare generally appropriate. Antibody-enzyme conjugates may be preparedusing a variety of coupling techniques (for review see, e.g., Scouten,W. H., Methods in Enzymology 135:30-65, 1987). Spectroscopic methods maybe used to detect dyes (including, for example, colorimetric products ofenzyme reactions), luminescent groups and fluorescent groups. Biotin maybe detected using avidin or streptavidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic, spectrophotometric or other analysis of thereaction products. Standards and standard additions may be used todetermine the level of Hsp70B′ polypeptide in a sample, using well knowntechniques.

As noted above, the ability to generate anti-Hsp70B′ antibodies todefined epitopes permits a variety of in vitro and in vivo uses. Forexample, anti-Hsp70B′ antibodies may be used to monitor the proteinlevels of a specific, sensitive, native, biomarker (Hsp70B′) in in vitrobioassays using human cell lines to evaluate the toxicity of chemicalcompounds. Using antibodies to defined epitopes on the Hsp70B′ andHsp70B proteins allows the specific monitoring of these proteins. Otheruses include the evaluation, diagnosis, prognosis and continuedmonitoring of specific disease conditions such as: hypertension,oncology, organ transplantation, ischaemia and trauma, infectioninflammation and fever, heart disease, autoimmune disorders,neurodegenerative diseases, monitoring spinal cord injuries,neuro-psychology evaluations, and chronic disease states.

An advantage of the present approach is that one can monitorperturbations in homeostasis by monitoring the levels of Hsp70B′ invivo. The Hsp70B′ protein can also be used as a biomarker in the fieldsof organ transplantation and cytoprotection. This would allow one toidentify physiological perturbations even when an individual has notreceived a definitive diagnosis with respect to a specific condition orto monitor patients exhibiting symptoms of known or unknown cause (e.g.,chronic fatigue syndrome). Such monitoring would help healthcareprofessionals gauge the severity of a condition, follow the progress ofthe patient, and decide when intervention may be needed. Evaluating astress response (made manifest by Hsp70B′ expression) may also be usefulin the care of patients who are in remission from an autoimmune, chroniccondition or neoplasia; to evaluate amniotic fluids or samples of theplacenta; and to assess newborn infants who are at risk (due, forexample, to premature birth). Monitoring Hsp70B′ is also useful inevaluating the fitness of healthy individuals (e.g., it can be used toassess training programs for high performance athletes). In yet otherapplications, the compositions and methods of the invention can be usedto evaluate the ability of various therapeutic compounds to oppose thestress condition, and in conjunction with in vivo diagnostic imaging(e.g., to evaluate wounding, inflammation or pathology in patients).

The following examples illustrate, not limit, the invention.

EXAMPLES

The following materials were obtained from the commercial suppliersindicated: anisole (Cat. No. A4405, Sigma Chemical Co., herein “Sigma”,St. Louis, Mo.); 2,2 ′-azino-di-(3-ethyl-benzthiazoline-sulfonic acid)(ABTS) (Cat. No. A6499, Molecular Probes Eugene, Oreg.); activatedMaleimide Keyhole Limpet Cyanin (Cat. No. 77106, Pierce Chemical Co.Rockford, Ill.); Biotin (Cat. No. B2643, Sigma); boric acid (Cat. No.B0252, Sigma); Sepharose® 4B (Cat. No. 17-0120-01, LKB/Pharmacia,Uppsala, Sweden); bovine serum albumin (LP) (Cat. No. 100 350,Boehringer Mannheim, Indianapolis, Ind.); cyanogen bromide (Cat. No.C6388, Sigma, St. Louis, Mo.); dialysis tubing Spectra/Por™ membraneMWCO: 6-8,000 (Cat. No. 132 665, Spectrum Industries Inc., Laguna Hills,Calif.); dimethyl formamide (DMF) (Cat. No. 22705-6, Aldrich ChemicalCompany, Milwaukee, Wis.); DIC (Cat. No. BP 592-500, Fisher);ethanedithiol (Cat. No. 39,802-0, Aldrich Chemicals, Milwaukee, Wis.);ether (Cat. No. TX 1275-3, EM Sciences); ethylenediaminetetraacetaticacid (EDTA) (Cat No. BP 120-1, Fisher Scientific, Springfield, N.J.);1-ethyl-3-(3′dimethylaminopropyl)-carbodiimide, HCL (EDC) (Cat No.341-006, Calbiochem, San Diego, Calif.); freund's adjuvant, complete(Cat. No. M-0638-50B, Lee Laboratories, Grayson, Ga.); freund'sadjuvant, incomplete (Cat. No. M0639-50B, Lee Laboratories); frittedchromatography columns (Column part No. 12131011; Frit: Part No.12131029, Varian Sample Preparation Products, Harbor City, Calif.);gelatin from bovine skin (Cat. No. G9382, Sigma); glycine (Cat. No.BP381-5, Fisher); goat anti-rabbit IgG, biotinylated (Cat No. A 0418,Sigma); HOBt (Cat. No. 01-62-0008, Calbiochem-Novabiochem); horseradishperoxidase (HRP) (Cat. No. 814 393, Boehringer Mannheim);HRP-Streptavidin (Cat. No. S 5512, Sigma); hydrochloric acid (Cat No.71445-500, Fisher); hydrogen peroxide 30% w/w (Cat. No. H1009, Sigma);methanol (Cat. No. A412-20, Fisher); microtitre plates, 96 well (Cat.No. 2595, Coming-Costar Pleasanton, Calif.); N-α-Fmoc protected aminoacids (Calbiochem-Novabiochem, San Diego, Calif.; see 1997-1998 catalogpages 1-45); N-α-Fmoc protected amino acids attached to Wang Resin(Calbiochem-Novabiochem; see 1997-1998 catalog pages 161-164); NMP (Cat.No. CAS 872-50-4, Burdick and Jackson, Muskegon, Mich.); peptide(Synthesized by Research Genetics, Inc., see below); piperidine (Cat.No. 80640, Fluka, available through Sigma); sodium bicarbonate (Cat. No.BP328-1, Fisher); sodium borate (Cat. No. B9876, Sigma); sodiumcarbonate (Cat. No. BP357-1, Fisher); sodium chloride (Cat. No. BP358-10, Fisher); sodium hydroxide (Cat. No. SS 255-1, Fisher);streptavidin (Cat. No. 1 520, Boehringer Mannheim); thioanisole (Cat.No. T-2765, Sigma); trifluoroacetic acid (Cat. No. TX 1275-3, EMSciences); Tween-20 (Cat. No. BP 337-500, Fisher); andWetbox-(Rubbermaid Rectangular Servin' Saver™ Part No. 3862 Wooster,Ohio).

The following general solutions were prepared: (1) BBS—Borate BufferedSaline with EDTA dissolved in distilled water (pH 8.2 to 8.4 with HCl orNaOH), which contains 25 mM Sodium borate (Borax), 100 mM Boric Acid, 75mM NaCl, and 5 mM EDTA; (2) 0.1 N HCl in saline, which containsconcentrated HCl (8.3 mL/0.917 L distilled water) and 0.154 M NaCl; (3)Glycine (pH 2.0 and pH 3.0) dissolved in distilled water and adjusted tothe desired pH, which contains 0.1 M glycine and 0.154 M NaCl; (4) 5×Borate 1× Sodium Chloride dissolved in distilled water, which contains0.11 M NaCl, 60 mM sodium borate, and 250 mM boric acid; and (5)substrate buffer in distilled water adjusted to pH 4.0 with sodiumhydroxide, which contains 50 to 100 mM Citric Acid.

The following peptide synthesis solutions were prepared: (1) AAsolution, in which HOBt is dissolved in NMP (8.8 grams HOBt to 1 literNMP) and Fmoc-N-a-amino is added at a concentration at 0.53 M; (2) DICsolution, which is 1 part DIC to 3 parts NMP; (3) a deprotectingsolution, which is 1 part piperidine and 3 parts DMF; and (4) Reagent R,which is 2 parts anisole, 3 parts ethanedithiol, 5 parts thioanisole,and 90 parts trifluoroacetic acid.

The following equipment was employed: an MRX plate reader (DynatechInc., Chantilly, Va.); a Hamilton Eclipse (Hamilton Instruments, Reno,Nev.); a Beckman TJ-6 centrifuge, refrigerated (Model No. TJ-6, BeckmanInstruments, Fullerton, Calif.); a Chart Recorder (Recorder 1 Part No.18-1001-40, Pharmacia LKB Biotechnology); a UV monitor (Uvicord SII PartNo. 18-1004-50, Pharmacia LKB Biotechnology); an Amicon Stirred CellConcentrator (Model 8400, Amicon Inc., Beverly, Mass.); 30 kDa MWcut-off filters (Cat. No. YM-30 Membranes Cat. No.13742, Amicon Inc.,Beverly, Mass.); a multi-channel automated pipettor (Cat. No. 4880,Coming Costar Inc., Cambridge, Mass.); a pH meter (Corning 240; CorningScience Products, Corning Glassworks, Corning, N.Y.); an ACT396 peptidesynthesizer (Advanced ChemTech, Louisville, Ky.); a vacuum dryer (Box isfrom Labconco, Kansas City, Mo.; Pump is from Alcatel, Laurel Md.); alyophilizer (Unitop 600 sl in tandem with Freezemobile 12, both fromVirtis, Gardiner, N.Y.).

Methods: Hsp70B′ Antibodies were produced as follows. Hsp70B′ antibodieswere produced in rabbits, goats and mice with either synthetic peptidesor recombinant Hsp70B′ protein as immunogen. Eight peptides were chosenfrom the human Hsp70B′ amino acid sequence. One of the Hsp70B′ peptides,the NT peptide MQAPRELAVGID(C) (SEQ ID NO:9) corresponded to anN-terminal fragment. The other seven fragments were derived from theC-terminal half of the Hsp70B′ protein and included the CC peptide(AHVFHVKGSLQEES; SEQ ID NO:7), the CA peptide (RDKIPEEDRRKMQ; SEQ IDNO:5), the CD peptide (RDKIPEEDRRKMQDKC; SEQ ID NO:4); the CB peptide(CGTQARQGDPSTGPI; SEQ ID NO:2), the ECB peptide (VPGGSSCGTQARQGDPSTGPI;SEQ ID NO:1), the TCB peptide (CGTQARQGDPST; SEQ ID NO:3), and the CEpeptide (GSLQEESLRDKIPEE; SEQ ID NO:10). The CB peptide was alsoresynthesized on a separate occasion and designated CB2. All peptideswere chemically coupled to KLH and animals were immunized with thepeptide conjugates. Recombinant human Hsp70B′ protein was purified to˜90% homogeneity and was also used as an immunogen. Primaryimmunizations were administered in Freund's complete adjuvant andsubsequent boosts were made in Freund's incomplete adjuvant. Animalswere immunized and boosted on a monthly basis. Sera were collected atvarious time points and the antibody response to the immunizing proteinor peptide was evaluated in an indirect enzyme immunoassay (EIA). Titreswere established as the dilution factor at which the absorbance in thetest sample was equal to 0.2 optical density units. In some instances,high-titre antisera from each set of animals were pooled and theantigen-specific antibody purified on peptide immunoaffinity columns.

Peptide was synthesized as follows. The event procedures included anincubation step (which allowed resin to be immersed in an appropriatesolution; all incubation steps occurred with mixing, a wash (addition of2 mls of DMF, incubation for 5 minutes and removal of the washsolution), and a wash cycle (consisting of 5 washes). For machinesynthesis, sequences of peptide were added to the peptide synthesizer.The C-terminal residue was determined and the appropriate Wang Resin wasattached to the reaction vessel. The peptides were synthesized from theC-terminus to the N-terminus by adding one amino acid at a time duringthe synthesis cycles. The amino acid residue selected for addition tothe peptide is controlled by sequence of the peptide that was enteredinto the database of the synthesizer.

The synthesis per se included a first step in which resin swelled (2 mlof DMF was added for 30 minutes and then removed), a second step inwhich the peptide was deprotected (1 ml deprotecting solution is addedto the reaction vessel and incubated for 20 minutes), washed, andcoupled (750 ml of amino acid solution and 250 ml of DIC solution areadded to the reaction vessel for 30 minutes and then washed out. Thecoupling step is repeated once before another wash cycle. The secondstep is repeated over the length of the peptide, with the amino acidsolution changing as the sequence listed in the peptide synthesizer'sdatabase dictates. In a third step, final deprotection occurs (thedeprotection and washing that occurs during the synthesis cycle areperformed one last time). Resins are deswelled in methanol by two rinseswith 5 ml methanol, a 5 minute incubation in 5 ml methanol, and a rinsein 5 mL methanol, and then vacuum dried.

Peptide was removed from the resin by incubation for 2 hours in reagentR and then precipitated into ether. The peptide was then washed inether, vacuum dried, resolubilized in diH₂0, frozen, and lyophilizedovernight. At this point, the peptide can be conjugated to KLH asfollows. The peptide (6 mg) is dissolved in PBS (6 ml) and mixed with 6mg of maleiimide activated KLH carrier in 6 ml of PBS for a total volumeof 12 mL. The entire solution was mixed for two hours, dialyzed in 1liter of PBS, and lyophilized.

Animals were immunized with peptide conjugates as follows. Three NewZealand White rabbits were injected in three to four subcutaneous dorsalsites with 250 μg of peptide-KLH conjugate in Freund's completeadjuvant. Booster shots (100 μg) were administered in Freund'sincomplete adjuvant. The total volume of each injection was 1 ml.

The rabbit immunization schedule was as follows: at Day 0 a “pre-immunebleed” was performed and the primary immunization was given; at week 2 afirst booster was given; at week 4 a blood sample was obtained; at week6 a second booster was given; at week 8 a second blood sample wasobtained and a third booster was given; at week 10 a third blood samplewas obtained; at week 12 a fourth booster was given; and at week 14 afourth and final blood sample was obtained.

Goats were injected with the same dose of peptide conjugate in Freund'sadjuvant as the rabbits received. The immunization schedule was also thesame except the booster at week 12 was not given and no blood sample wastaken at week 14.

BALB/c mice were immunized intraperitoneally with 50 μg peptideconjugate in Freund's complete adjuvant on day 0, and in Freund'sincomplete adjuvant at weeks 2, 5 and 8. Mouse test bleeds werecollected on week 7 and 10.

Rabbits were also immunized with recombinant human Hsp70B′ protein. Morespecifically, three New Zealand White rabbits were immunized on day 0with purified recombinant human Hsp70B′ protein in Freund's completeadjuvant. Booster shots (100 μg) were given at weeks 3, 4, 6, and 8, andblood samples were collected at weeks 5, 7, 9 and 10. The blood sampleswere processed for serum, and the antibody obtained was designated 70B′WP.

To collect rabbit serum, the rabbits were bled (30 to 50 ml) from theauricular artery. The blood was allowed to clot at room temperature for15 minutes and the serum was separated from the clot using an IECDPR-6000 centrifuge at 5000×g. Cell-free serum was decanted gently intoa clean test tube and stored at −20° C. for affinity purification.

Anti-peptide titre was determined as follows. All solutions were addedby a liquid handling dispenser (the Hamilton Eclipse), with theexception of the wash solution. The anti-peptide titres in the rabbits,goats, and mice were determined in an ELISA with peptide on the solidphase. Flexible high binding ELISA plates were passively coated withpeptide diluted in BBS (100 μL, 1 μg/well) and the plate was incubatedat 4° C. in a wetbox overnight (air-tight container with moistenedcotton balls). The plates were emptied and then washed three times withBBS containing 0.1% Tween-20 (BBS-TW) by repeated filling and emptyingusing a semi-automated plate washer. The plates were blocked bycompletely filling each well with BBS-TW containing 1% BSA and 0.1%gelatin (BBS-TW-BG) and incubating for 2 hours at room temperature. Theplates were emptied and sera of both pre- and post-immune serum wereadded to wells. The first well contained sera at 1:50 in BBS. The serawere then serially titrated eleven more times across the plate at aratio of 1:1 for a final (twelfth) dilution of 1:204,800. The plateswere incubated overnight at 4° C. The plates were emptied and washedthree times as described.

Biotinylated secondary antibodies (100 μl) were added to each microtitreplate test well and incubated for four hours at room temperature. Theplates were emptied and washed three times. Horseradishperoxidase-conjugated Streptavidin (100 μL diluted 1:10,000 inBBS-TW-BG) was added to each well and incubated for two hours at roomtemperature. The plates were emptied and washed three times. The ABTSwas prepared fresh from stock by combining 10 mL of citrate buffer (0.1M at pH 4.0), 0.2 mL of the stock solution (15 mg/mL in water) and 10 μLof 30% H₂O₂. The ABTS solution (100 μL) was added to each well andincubated at room temperature. The plates were read at 414 λ, 20 minutesfollowing the addition of substrate. Titres were established as thereciprocal dilution factor at which the test sample was equal to 0.2absorbance units.

The Anti-Hsp70B′ protein titre was determined by indirect ELISA withHsp70B′ on the solid phase. Nunc Maxisorp ELISA plates were passivelycoated with purified recombinant human Hsp70B′ diluted in PBS (100 μl,100 ng/well) at 4° C. overnight. Plates were washed six times with PBScontaining 0.05% tween-20 (Bio-Rad) and then blocked at room temperaturewith 200 μl/well blotto (5% non-fat milk (Carnation), 0.05% tween-20,0.02% thimerosal (Fisher Scientific) in PBS). Plates were washed and theantiserum, diluted 1:1000 in blotto, was added to the wells. The dilutedantiserum was serially titrated 5 times at a ratio of 1:3 in blotto fora final dilution of 1:243000. Plates were incubated at room temperaturefor 1hour, followed by washing as described. Peroxidase conjugatedanti-rabbit IgG (100 μl/well of SAB-300, Stressgen Biotechnologies),diluted 1:25000 in blotto, was added to the wells and the plates wereincubated for 1 hour at room temperature. Plates were washed asdescribed and then developed with tetramethylbenzadine (TMB; BioFx) for5 -10 minutes at room temperature. Color development was stopped by theaddition of acid stop solution (BioFx). Absorbance of each well wasmeasured at 450 nm in an EL808 microplate reader (BioTek), interfacedwith KC3 software. Titres were established as the reciprocal dilutionfactor at which the test sample was equal to 0.2 absorbance units.

The peptide affinity purification column was prepared by conjugating 5mg of peptide to 10 ml of cyanogen bromide-activated Sepharose 4B, and 5mg of peptide to hydrazine-Sepharose 4B. Briefly, 100 uL of DMF wasadded to peptide (5 mg) and the mixture was vortexed until the contentswere completely wetted. Water was then added (900 μL) and the contentswere vortexed until the peptide dissolved. Half of the dissolved peptide(500 μL) was added to separate tubes containing 10 mL ofcyanogen-bromide activated Sepharose 4B in 0.1 mL of borate bufferedsaline at pH 8.4 (BBS), and 10 mL of hydrazine-Sepharose 4B in 0.1 Mcarbonate buffer adjusted to pH 4.5 using excess EDC in citrate bufferpH 6.0. The conjugation reactions were allowed to proceed overnight atroom temperature. The conjugated Sepharose was pooled and loaded ontofritted columns, washed with 10 mL of BBS, blocked with 10 mL of 1 Mglycine, and washed with 10 mL 0.1 M glycine adjusted to pH 2.5 with HCland re-neutralized in BBS. The column was washed with enough volume forthe optical density at 280λ to reach baseline.

To affinity purify antibodies, the peptide affinity column was attachedto a UV monitor and chart recorder. The titred rabbit antiserum wasthawed and pooled. The serum was diluted with one volume of BBS andallowed to flow through the columns at 10 mL per minute. The non-peptideimmunoglobulins and other proteins were washed from the column withexcess BBS until the optical density at 280 λ reached baseline. Thecolumns were disconnected and the affinity purified column was elutedusing a stepwise pH gradient from pH 7.0 to pH 1.0. The elution wasmonitored at 280 nM, and fractions containing antibody (pH 3.0 to pH1.0) were collected directly into excess 0.5 M BBS. Excess buffer (0.5 MBBS) in the collection tubes served to neutralize the antibodiescollected in the acidic fractions of the pH gradient.

The entire procedure was repeated with “depleted” serum to ensuremaximal recovery of antibodies. The eluted material was concentratedusing a stirred cell apparatus and a membrane with a molecular weightcutoff of 30 kD. The concentration of the final preparation wasdetermined using an optical density reading at 280 nM. The concentrationwas determined using the following formula: mg/mL=OD₂₈₀/1.4.

Antibody titres were determined by indirect EIA, essentially as alreadydescribed for the anti-peptide and anti-Hsp70B′ antibodies. Briefly,Nunc Maxisorp ELISA plates were passively coated with 100 ng/well ofrecombinant human Hsp70B′, recombinant human Hsp70A (SPP-755, StressgenBiotechnologies), recombinant bovine Hsc70 (SPP-751, StressgenBiotechnologies), recombinant hamster Grp78 (SPP-765, StressgenBiotechnologies), E. coli DnaK (SPP-630, Stressgen Biotechnologies) andrecombinant M. tuberculosis Hsp71 (Stressgen Biotechnologies) diluted inPBS. Similarly, unconjugated CB2, ECB, TCB, and CE peptides were dilutedin PBS and coated at 0.5 μg/well. Plates were blocked with 200 μl/wellof casein blocking buffer in PBS (Pierce) for 2 hours at roomtemperature. Test and negative control antibodies were diluted to astarting concentration/dilution of 1 μg/ml for affinity purifiedantibodies and 1:1000 for serum antibodies in Stabilzyme Select(SurModics). Diluted antibodies were added to the plate wells and werefurther diluted by 5 serial titrations at a ratio of 1:3 to a finalconcentration/dilution of 4.1 ng/ml (for purified antibodies) and1:243000 (for serum antibodies). Plates were incubated for 1 hour atroom temperature with diluted primary antibody, followed by another 1hour room temperature incubation with peroxidase conjugated anti-rabbitIgG (SAB-300, Stressgen Biotechnologies), diluted 1:25000 in StabilzymeSelect. Plates were developed with TMB (BioFx) for 5 -10 minutes at roomtemperature and the reaction was stopped with acid stop solution(BioFx). Absorbance of each well was measured at 450 nm in an EL808microplate reader (BioTek), interfaced with KC3 software. The titre ofthe antibody was represented as the concentration or reciprocal dilutionof the antibody that resulted in an absorbance reading of 0.2.

A relative titre index was established to compare the titre of the testantibody against a negative control antibody. For affinity purifiedantibodies, the index value was calculated by dividing the titre of thenegative control antibody by the titre of the test antibody. For serumantibodies, the index value was calculated by dividing the titre of thetest antibody by the titre of the negative control antibody.

Competition EIA. Nunc Maxisorp ELISA plates (primary plates) werepassively coated overnight at 4° C. with 100 ng/well of purifiedrecombinant human Hsp70B′ diluted in PBS. After washing six times withPBS containing 0.05% tween-20, primary plates were incubated with 200μl/well of Superblock blocking buffer in PBS (Pierce) at roomtemperature.

While the primary plates were blocking, the free antigen:antibodymixtures were prepared in 96 well Nunc polypropylene plates (secondaryplates). First, Hsp70B′ standard and Hsp70 protein homologs (i.e. crossreactants; recombinant human Hsp70A, recombinant bovine Hsc70 ,recombinant hamster Grp78, E. coli DnaK, and M. tuberculosis Hsp71) werediluted to a starting concentration of 10 μg/ml and 500 μg/mlrespectively in BSA diluent (0.12% BSA (Sigma), 0.05% tween-20 (BioRad),1:1000 ProClin 200 (Supelco) in PBS). The diluted proteins were added tothe secondary plates and then serially titrated four times at a ratio of1:5 in BSA diluent to generate a Hsp70B′ concentration range of 0.016-10μg/ml and a homolog concentration range of 0.8-500 μg/ml in a finalvolume of 100 μl/well. BSA diluent alone served as the 0 μg/ml point.

The rabbit CB2 affinity purified antibody, CE and 70B′ WP serumantibodies were diluted to 0.06 μg/ml, 1:10,000 and 1:16,000respectively in BSA diluent. An equal volume (100 μl/well) of dilutedantibody was added to the secondary plate wells with BSA diluent andvarying concentrations of Hsp70B′ and homolog. The secondary plate wasincubated at room temperature for 1.75 hours, after which 100 μl of thefree antigen:antibody mixture was transferred to the blocked and washedprimary plates. Primary plates were incubated at room temperature for 1hour at room temperature, followed by a 1 hour room temperatureincubation with peroxidase conjugated anti-rabbit IgG secondary antibody(SAB-300, Stressgen Biotechnologies) diluted in BSA diluent. The primaryplate was developed with TMB (BioFx) and stopped with acid stop solution(BioFx) after 10 minutes. The absorbance of each well was read with aBioTek EL808 microplate reader set at 450 nm. The maximum absorbance(i.e. A_(max); maximum amount of antibody binding at 0 μg/ml freeHsp70B′ or homolog) was determined and used to calculate the % A/A_(max)at each Hsp70B′ or homolog competition concentration. Antibodydisplacement curves were generated by plotting the % A/A_(max) againstthe concentration of free Hsp70B′ or homolog.

Cloning and Expression of Recombinant Human Hsp70B′. Human Hsp70B′ wascloned from heat shocked HeLa cells and expressed recombinantly in E.coli. Briefly, 2×10⁷ HeLa cells were heat shocked for 2 hours at 44° C.and then immediately harvested. Poly (A+) RNA was isolated from the heatshocked HeLa cells with a mRNA isolation kit (Boehringer Mannheim) andused to synthesize human Hsp70B′ cDNA by RT-PCR. The 51 μl RT-PCRreaction mixture consisted of 1 μl of 10 mM dNTP (Perkin Elmer), 2.5 μlof 100 mM DTT (Boehringer Mannheim), 0.25 μl of 40 units/μl RNaseinhibitor (Boehringer Mannheim), 10 μl of 5× RT-PCR buffer containing7.5 mM MgCl₂ and DMSO (Boehringer Mannheim), 1 μl of enzyme mix(Boehringer Mannheim) containing Expand High Fidelity enzyme mix and AMVreverse transcriptase, 0.87 μg of poly (A+) RNA from heat shocked HeLacells, and 1 μg each of: primer1:5′-GAAGCTTCACATATGCAGGCCCCACGGGAGCTCG-3′ (SEQ ID NO:12) and primer2:5′-GAAGCTCGAGTCAATCAACCTCCTCAATGA-3′ (SEQ ID NO: 13).

The primer sequences were derived from the human Hsp70B′ nucleotidesequence (Leung et al., 1990) and designed to introduce a NdeIrestriction site upstream of the start codon and a XhoI restriction sitedownstream of the stop codon. The cDNA was synthesized by incubating thereaction mixture for 30 minutes at 50° C. and amplified by PCR in aPerkin Elmer Gene Amp PCR System 2400 which was programmed for 2 minutesat 94° C., followed by 10 cycles of 30 seconds at 94° C., 30 seconds at60° C., 2 minutes at 68° C., and 15 cycles of 30 seconds at 94° C., 30seconds at 60° C., and 2.5 minutes at 68° C.; the last extension step of7 minutes at 68° C. The reaction product was analyzed by agarose gelelectrophoresis and ethidium bromide staining. The human Hsp70B′ cDNAwas digested with NdeI (New England BioLabs) and XhoI (New EnglandBioLabs) and ligated into a similarly digested pET24a (Novagen)expression vector. The resulting expression plasmid carrying humanHsp70B′ was transformed into E. coli BLR (DE3) and BL21 (DE3) cells. Forpreparation of bacterial extracts, cells were grown at 37° C. in LBmedium (Difco) containing 30 μg/ml kanamycin (Sigma). When the cellsreached an OD₅₉₅ of 0.5, 1 mM isopropyl β-D-thiogalactopyranoside(Amersham) was added to the medium and the culture was incubated at 37°C. for an additional 2.5 hours. Cell pellets were resuspended with lysisbuffer (50 mM Tris pH 7.5 (BDH), 150 mM NaCl (BDH), 0.1 mMphenylmethylsulfonyl fluoride (Sigma), 1 μg/ml leupeptin (Sigma) and 1μg/ml aprotinin (Sigma)) and disrupted by sonication. The Bradford assay(Bio-Rad) was used to determine the protein concentration of theextract. The extract was diluted in SDS-PAGE sample buffer (40 mMTris-HCl pH 6.8 (BDH), 1% SDS (Bio-Rad), 50 mM DTT (ICN), 7.5% glycerol[Anachemia], and 0.003% bromophenol blue (Sigma)) and boiled for 5minutes in preparation for SDS-PAGE and immunoblotting.

Cloning and Expression of Recombinant His₆-Human Hsp70B (Fragment). The741bp fragment that encodes a portion of the amino terminus region ofthe human Hsp70B was obtained from SPD-925, a human Hsp70B stress geneprobe (Stressgen Biotechnologies). SPD-925 is supplied as a plasmidcontaining 3.15 kb of the 5′ non-transcribed Hsp70B gene sequence, the119 bp RNA leader region and the 741 bp protein coding region. Althoughthe protein coding region can be excised from SPD-925 by digestion withHindIII, restriction site modifications were introduced by PCR. The 50μl PCR reaction mixture consisted of 8 μl of 1.25 mM dNTP (New EnglandBioLabs), 5 μl of 10× Expand High Fidelity PCR buffer (BoehringerMannheim), 0.5 μl of 3.5 units/μl Expand High Fidelity DNA polymerase(Boehringer Mannheim), 0.05 μg of SPD-925, and 1 μg each of primer 1:

5 ′-GAAGCTTCACATATGCAGGCCCCACGGGAGCTCG-3′ (SEQ ID NO:12) and primer 25′-TGACAAGCTTAGAATTCTTCCATGAAGTGGT-3′ (SEQ ID NO:14).

The primer sequences were derived from the human Hsp70B nucleotidesequence (Voellmy et al., 1985). Primer 1 was designed to introduce anNdeI restriction site upstream of the start codon whereas primer 2 wasdesigned to introduce a stop codon and a HindIII restriction site. ThePCR reaction was performed in a Perkin Elmer Gene Amp PCR System 2400which was programmed for 2 minutes at 94° C., followed by 10 cycles of30 seconds at 94° C., 30 seconds at 60° C., 1 minute at 72° C., and 15cycles of 30 seconds at 94° C., 30 seconds at 60° C., and 1.25 minutesat 72° C.; the last extension step was 7 minutes at 72° C. The reactionproduct was analyzed by agarose gel electrophoresis and ethidium bromidestaining. The human Hsp70B PCR product was digested with NdeI (NewEngland BioLabs) and HindIII (New England BioLabs) and ligated into asimilarly digested pET28a (Novagen) expression vector. The resultingexpression plasmid carrying his₆-human Hsp70B was transformed into E.coli BLR (DE3) cells. For preparation of bacterial extracts, cells weregrown at 37° C. in LB medium (Difco) containing 30 μg/ml kanamycin(Sigma). When the cells reached an OD₅₉₅ of 0.5, 1 mM isopropylβ-D-thiogalactopyranoside (Amersham) was added to the medium and theculture was incubated at 37° C. for an additional 2.5 hours. Cellpellets were resuspended with SDS-PAGE sample buffer, sonicated, andboiled for 5 minutes in preparation for SDS-PAGE and immunoblotting.

Purification of Recombinant Human Hsp70B′ Protein. E. coli BL21 (DE3)cells harboring the expression plasmid for full length human Hsp70B′were grown in LB media with 30 μg/ml kanamycin (Sigma) at 37° C. withshaking at 250 rpm. At an OD₅₉₅nm of 0.5-0.6, expression of recombinantprotein was induced with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG;Calbiochem) and cells were harvested by centrifugation after 3 hours.Bacterial pellets were resuspended with lysis buffer (25 mM Tris-HCl pH7.5 (BDH), 5 mM EDTA (Sigma), 0.3 mg/ml lysozyme (Sigma), 5 mMp-aminobenzamidine (Sigma), 15 mM β-mercaptoethanol (BME; Sigma), 1 mMphenylmethylsulfonyl fluoride (PMSF; Sigma), 1 μg/ml aprotinin (Sigma),1 μg/ml leupeptin (Sigma)) and disrupted by sonication. The bacterialsuspension was centrifuged and the supernatant was loaded onto a Qsepharose (Amersham Pharmacia) ion exchange column. Bound protein waseluted with a 0 -300 mM NaCl gradient, followed by a 300 mM NaCl wash.Q-Sepharose fractions were analyzed on coomassie stained SDS-PAGE gelsand the Hsp70B′ containing fractions pooled. MgCl₂ (Sigma) was added toa final concentration of 3 mM and the pool was loaded onto an ATPagarose (Fluka) column. After 500 mM NaCl and 20 mM NaCl washes, boundHsp70B′ protein was eluted with ATP agar elution buffer (10 mM Tris-HClpH 7.5, 3 mM MgCl₂, 10 mM ATP (Sigma), 15 mM BME, 0.1 mM PMSF, 1 μg/mlaprotinin, 1 μg/ml leupeptin). Hsp70B′ containing fractions were pooledand sodium acetate (Mallinckrodt) pH 5.5 added to a final concentrationof 40 mM. The pool was adjusted to pH 5.5 with acetic acid (FisherScientific) and then loaded onto a SP HiTrap (Amersham Pharmacia) ionexchange column. Protein was eluted with a 0 -1 M NaCl gradient. Hsp70B′containing fractions were pooled and the protein preparation wasdialyzed and stored in 10 mM Tris-HCl pH 7.5, 150 mM NaCl buffer.Protein concentration was determined by the Bradford assay (Bio-Rad) andpurity was assessed by densitometry scanning of a coomassie stained SDSgel with 0.5, 1.0 and 1.5 μg Hsp70B′.

Cell Culture was performed as follows. HeLa (human epitheloid cervicalcarcinoma, ATCC CCL-2) cells were grown in Eagle's minimal essentialmedium with Earle salts (ICN) supplemented with 0.1 mM non-essentialamino acids (Gibco), 2 mM L-glutamine (Gibco), 10% fetal bovine serum(Gibco), 50 μg/ml gentamycin sulfate (Gibco), and 1 mM sodium pyruvate(Gibco). Jurkat (clone E6-1, human acute T-cell leukemia, ATCC TIB-152)cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetalbovine serum (Gibco), 2 mM L-glutamine (Gibco), 1.5 g/L sodiumbicarbonate (ICN), 10 mM HEPES (Gibco), 4.5 g/L glucose (Sigma), 1 mMsodium pyruvate (Gibco), and 50 μg/ml gentamycin sulfate (Gibco), and1.5 g/L sodium bicarbonate (ICN). Vero (African green monkey, normalkidney epithelial, ATCC CCL-81) cells were grown in Eagle's minimalessential medium with Earle salts (ICN) supplemented with 2 mML-glutamine (Gibco), 0.1 mM non-essential amino acids (Gibco), 1 mMsodium pyruvate (Gibco), 0.1 mM non-essential amino acids, 50 μg/mlgentamycin sulfate (Gibco) and 10% fetal bovine serum (Gibco). CHO-K1(Chinese hamster ovary epithelial, ATCC CCL-61) cells were grown inDulbecco's modified Eagle's medium (Gibco) supplemented with 2 mML-glutamine, 10 mM HEPES (Gibco), 50 μg/ml gentamycin sulfate (Gibco)and 5% fetal bovine serum (Gibco). MDBK (bovine normal kidneyepithelial, ATCC CCL-22) cells were grown in Eagle's minimal essentialmedium with Earle salts (ICN) supplemented with 2 mM L-glutamine(Gibco), 0.1 mM non-essential amino acids (Gibco), 1 mM sodium pyruvate(Gibco), 50 μg/ml gentamycin sulfate (Gibco) and 10% horse serum(Gibco). HeLa, Jurkat, Vero, CHO and MDBK cells were incubated at 37° C.in a water-jacketed incubator with 5% CO₂. A-431 cells were similarlyincubated at 37° C., but with 10% CO₂.

Treatment with Metals, Azetidine and Heat. At 90% confluency, HeLa,A-431 and Jurkat cells were incubated at 37° C. for 2 hours with 100 μMCdCl₂ (Sigma) or 250 μM ZnCl₂ (Sigma) and harvested after a 5 hourrecovery period in media without CdCl₂ or ZnCl₂. Cells were similarlytreated for 5 hours with 5 mM of the proline analogue,L-azetidine-2-carboxylic acid (Sigma), and harvested after a 2 hourrecovery period. Cells were also heat shocked for 20 minutes or 2 hoursat 44° C. and harvested after a 5 hour recovery period at 37° C. Celllysates were prepared for SDS polyacrylamide gel electrophoresis andimmunoblot analysis.

Vero, CHO and MDBK cells were also subjected to heat stress. Vero cellswere heat stressed for 1.5 hours at 42° C. and recovered at 37° C. for18 hours prior to harvesting. CHO cells were heat stressed for 2 hoursat 42° C. and recovered at 37° C. for 18 hours prior to harvesting. MDBKcells were heat stressed for 1.5 hours at 44° C. and harvested after an18 hour recovery at 37° C. Cell lysates were prepared for SDSpolyacrylamide gel electrophoresis and immunoblot analysis.

Heat Treatment of HeLa Cells. HeLa cells were grown to 90% confluencyand heated for 2 hours at 37° C., 38.5° C., 40° C., 41.5° C., 43° C.,and 44.5° C. Cells were harvested after a 5 hour recovery period at 37°C. and prepared for SDS polyacrylamide gel electrophoresis andimmunoblot analysis.

Recovery of Heat Treated HeLa Cells. HeLa cells were grown to 90%confluency and heated for 2 hours at 44.5° C. Cells were harvested afterrecovering at 37° C. for 0, 2.5, 5, 16 and 24 hours. Control cells weremaintained at 37° C. and were similarly harvested at the same recoverytimes. Cell lysates were prepared for SDS polyacrylamide gelelectrophoresis and immunoblot analysis.

SDS Polyacrylamide Gel Electrophoresis and Immunoblotting. After theappropriate recovery period, cells were washed with Dulbecco's phosphatebuffered saline without calcium and magnesium (ICN) and harvested with acell scraper. Harvested cells were resuspended with lysis buffer(Dulbecco's phosphate buffered saline without calcium and magnesium(ICN), 0.05% triton-X100 (Sigma), 0.1 mM phenylmethylsulfonyl fluoride(Sigma), 1 μg/ml leupeptin (Sigma) and 1 μg/ml aprotinin (Sigma)) anddisrupted by sonication. The protein content of the cellular extractswas determined by the Bradford method (Bio-Rad). The extract was dilutedin SDS buffer containing 40 mM Tris-HCl pH 6.8 (BDH), 1% SDS (Bio-Rad),50 mM DTT (ICN), 7.5% glycerol (Anachemia), and 0.003% bromophenol blue(Sigma) and heated at 70° C. for 5 minutes. Cell extracts were resolvedon 12.5% SDS polyacrylamide gels and separated proteins wereelectroblotted onto nitrocellulose membranes (Gelman Sciences) by usinga 25 mM Tris ) (BDH), 192 mM glycine (Fisher Scientific), 20% (v/v)methanol (Fisher Scientific) transfer buffer in a Trans-Blot apparatus(Bio-Rad) at 100 V for 1 hour. Blots were blocked in 5% Carnationnon-fat milk, 0.05% Tween-20 (Bio-Rad), 0.02% NaN₃ (Fisher Scientific)or thimerosal (Fisher Scientific) in phosphate buffered saline (15.4 mMNa₂HPO₄ (Mallinckrodt), 4.6 mM NaH₂PO₄ (Mallinckrodt), 120 mM NaCl(BDH)). Blots were probed with a mouse monoclonal antibody specific forinducible Hsp70A and Hsp70B′ (Stressgen Biotechnologies, SPA-810),rabbit polyclonal antibody specific for inducible Hsp70A (StressgenBiotechnologies, SPA-812), rat monoclonal antibody specific for cognateHsc70 (Stressgen Biotechnologies, SPA-815), rabbit polyclonal antibodyfor Hsp110 (Stressgen Biotechnologies, SPA-1101), mouse monoclonalantibody specific for Grp75 (Stressgen Biotechnologies, SPA-825), mousemonoclonal antibody specific for the endoplasmic reticulum KDELretention signal peptide (Stressgen Biotechnologies, SPA-827), mousemonoclonal antibody specific for E. coli DnaK (StressgenBiotechnologies, SPA-880), mouse monoclonal antibody specific for M.tuberculosis Hsp71 (Stressgen Biotechnologies, SPA-885), and polyclonalantibodies raised either against a panel of synthetic peptides derivedfrom the linear human Hsp70B′ sequence or purified recombinant humanHsp70B′. Blots were incubated for 1 hour at room temperature withprimary antibodies diluted in blocking buffer. Alkaline phosphatase orperoxidase conjugated secondary antibodies (Stressgen Biotechnologies)were respectively diluted 1:1000 and 1:5000 in blocking buffer andincubated with the blots for 1 hour at room temperature. Blots usingalkaline phosphatase conjugated secondary antibodies were developed with0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (Sigma) and 0.3 mg/mlnitro blue tetrazolium (Sigma) in alkaline phosphatase buffer (100 mMTris-HCl pH 9.5 (BDH), 150 mM NaCl (BDH), 10 mM MgCl₂ (FisherScientific)). The developed blots were washed with deionized water tostop the colour reaction. Blots using peroxidase conjugated secondaryantibodies were developed by enhanced chemiluminescence (ECL, Amersham).

Generation of Mouse Hybridomas. BALB/c mice were immunized with Hsp70B′peptide-KLH conjugate essentially as already described. Test bleeds wereanalyzed for anti-peptide and anti-Hsp70B′ protein serum titres byindirect EIA and mice with high Hsp70B′ titres were selected for fusion.Four days prior to splenectomy and cell fusion, the selected mice wereboosted with antigen in the absence of adjuvant. Mouse spleens wereaseptically removed, minced with forceps and strained through a sieve.Cells were washed twice with IMDM medium and counted using ahemocytometer. Spleen cells were mixed with mouse myeloma P3×63Ag8.653cells at a ratio of 5:1 (spleen:myeloma cells) and centrifuged. Cellpellets were resuspended with 1 ml of 50% PEG (MW 1450), added dropwiseover a period of 30 seconds. The resuspended cells were gently mixed for30 seconds using a pipette and then allowed to stand undisturbed foranother 30 seconds. 5 ml of IMDM media was added over a period of 90seconds, followed immediately with another 5 ml. The resulting cellsuspension was left undisturbed for 5 minutes, after which the cellswere pelleted and resuspended at 5×10⁵ cells/ml in HAT medium (IMDMcontaining 10% FBS, 2 mM L-glutamine, 0.6% 2-mercaptoethanol,hypoxanthine, aminopterin, thymidine, and 10% Origen growth factor).Cells were plated into 96 well plates at 10⁵ cells/well. Plates wereincubated at 37° C. in a 7% CO₂ atmosphere with 100% humidity. Sevendays after fusion, the media was removed and replaced with IMDMcontaining 10% FBS, 2mM L-glutamine, 0.6% 2-mercaptoethanol,hypoxanthine and thymidine. 10-14 days after fusion, the supernatant wastaken from wells with growing hybridoma colonies and screened foranti-peptide and anti-protein antibody by indirect EIA. Hybridoma cellsfrom positive wells were cloned by limiting dilution in 96 well platesat a density of 0.25 cells per well or one cell in every fourth well.Growing colonies were tested 10-14 days later using the same assay(s)used to initially select the hybridomas. Positive clones were expandedand frozen.

Results

Eight peptide sequences were chosen, one an N-terminus epitope as wellas seven epitopes from the carboxyl end of the human Hsp70B′ protein.Peptide sequences were selected based on minimal identity and homologywith other HSP70 family members and algorithmic predictions ofhydrophilicity (Kyte-Doolittle), antigenicity (Jameson-Wolf) and surfaceprobability (Emini). These sequences, their respective residue numbers(according to the GeneBank sequence #P17066) and epitope designationsare listed in Table 1. The CB and CB2 sequences are the same, except theCB2 peptide was synthesized on a separate occasion.

TABLE 1 Summary of Immunogenicity of Different Hsp70B′ Epitopes Epitope(Ab) Location Anima Antisera Titer # Animals Name (P17066) PeptideImmunogen 1 Host (EIA) Responding *CB 624-638 KLH-CGTQARQGDPSTGPIRabbit >204,800 3/3 33,021 >204,800 *CA 561-573 KLH-(C)RDKIPEEDRRKMQRabbit >204,000 3/3 >204,800 191,311 *CD 561-576 RDKIPEEDRRKMQDKC-KLHRabbit >204,000 2/2 >204,800 *CC 546-559 KLH-AHVFHVKGSLQEESRabbit >204,000 3/3 >204,800 >204,800 *NT  1-12 MQAPRELAVGID(C)-KLHRabbit >204,000 3/3 168,019 >204,800 ECB 618-638KLH-vpggsscgtqarqgdpstgpi Rabbit >204800 3/3 >204800 >204800 TCB 624-635KLH-CGTQARQGDPST Rabbit >204800 3/3 >204800 >204800 CE 553-567KLH-GSLQEESLRDKIPEE Rabbit 8424 3/3 67510 4368 *CB2 624-638KLH-CGTQARQGDPSTGPI Rabbit >204800 3/3 >204800 >204800 CB2 624-638KLH-CGTQARQGDPSTGPI Goat 4200 1/1 CB2 624-638 KLH-CGTQARQGDPSTGPIMouse >204800 10/10 111432 >204800 >208400 36685 108253 151239 >20480064358 >204800 70B′ Hsp70B′ full length recombinant Rabbit >243000 3/3 WP  1-643 human Hsp70B′ protein 123100 222900

The peptides were synthesized, conjugated to KLH and used to immunizerabbits, goats or mice. Specific antibody responses to the immunizingpeptide were detected in every animal as assessed by indirect peptideEIA (Table 1). Six of the nine rabbit anti-peptide antisera were thenpurified by peptide immunoaffinity chromatography. Antibody preparationswere tested for specificity of binding with a panel of HSP70 proteinhomologs as well as a series of stressed and non-stressed human andmammalian cell lysates (Tables 2-6).

Hsp70B′ Antibody Production with Recombinant Human Hsp70B′ Protein.

In addition to generating Hsp70B′ antibodies via the peptideimmunization route, a Hsp70B′ antibody, designated 70B′ WP, was producedin rabbits using purified (i.e. ˜90% homogeneity) recombinant humanHsp70B′ as the immunogen. Like the peptide generated antibodies, theantibody response to the immunizing protein was detected in each rabbitby indirect EIA (Table 1). The sera was pooled and then tested forspecificity against a panel of purified HSP70 homologs by indirect EIA(Table 2) and competition EIA. (Table 3) The antisera was also assessedby immunoblotting with control and heat stressed cell lysates (Tables 5and 6).

Determination of Antibody Specificity by Relative Titre Index

The specificity of selected Hsp70B′ antibodies was determined byindirect EIA and expressed as an antibody titre index, relative to anirrelevant or negative control antibody (Table 2). Titre is defined asthe antibody concentration (for purified antibodies) or reciprocaldilution (for unpurified antisera) that results in 0.2 absorbance units.This cutoff value approximately represents the absorbance of the assaybackground+3 standard deviations. It is the lowest distinguishablepositive signal at 95% confidence.

The relative titre index directly compares the titres of the Hsp70B′antibodies with the titre of an irrelevant antibody. It is therefore ameasurement of antibody reactivity towards a particular protein orpeptide above non-specific binding. The index value is directlyproportional to the reactivity of the antibody. Thus, the higher therelative index, the more reactive the antibody is toward a particularprotein or peptide. An index of 1 reflects reactivity that is on parwith that of the negative control antibody and is considered asnegligible binding. An index value greater than 1 is indicative ofbinding. If an antibody is found to react with only one protein orpeptide (e.g. the antibody has an index of >1 with only one protein orpeptide), specificity can be defined within the context of the proteinsor peptides tested.

Based on absolute index values in Table 2, the CB2, CE, ECB and TCBantibodies preferentially reacted with Hsp70B′ by 177, 48, 41 and 29fold respectively over other HSP70 protein homologs. Under these assayconditions, the CB2, CE, ECB and TCB antibodies were specific forHsp70B′ protein.

At lower dilutions, the 70B′ WP antibody minimally exhibited some crossreactivity with Hsp70A, DnaK, and likely with Hsc70 and Hsp71. However,the 70B′ WP antibody still reacted 90 fold higher with Hsp70B′ overHsp70A and DnaK. SPA-812 is a rabbit polyclonal antibody produced torecombinant human Hsp70A protein. In addition to reacting with Hsp70A,SPA-812 also cross reacted with Hsp70B′ (and likely with Hsc70 and DnaK)at lower dilutions. For both of these whole protein antibodies,reactivity with their respective antigens was high (index >100).Therefore, by using the antibodies at higher dilutions, cross reactivitycan likely be “diluted out” and reactivity to the intended protein stillretained. This essentially selects for the population of higher affinityantibodies that recognize specific epitopes on the antigen of interest.

The 70B′ WP antibody reacted 4-6 fold higher with Hsp70B′ protein overthe CE, ECB and TCB serum antibodies. However, the 70B′ WP antibody alsoexhibited cross reactivity with other homologs whereas the peptideantibodies did not. This is not unexpected since the 70B′ WP antibodylikely consists of several antibody populations that recognize differentHsp70B′ epitopes throughout the surface of the molecule. Some of theseepitopes though are likely shared or homologous epitopes with otherhomologs. The peptide antibodies probably also consist of severalantibody populations that recognize Hsp70B′, but the epitopes arelimited to the immunizing peptide sequence, thus limiting crossreactivity. This supports the peptide approach over whole proteinimmunizations, if specific Hsp70B′ polyclonal antibodies are required.Reactivity of peptide polyclonal antibodies with the intended proteincan also be enhanced by affinity purification, as illustrated by thepurified CB2 antibody. Under these assay conditions, this antibody had ahigh index value (i.e. >100) for Hsp70B′ and was specific for Hsp70B′.Of course, the 70B′ WP could also be affinity purified to enhancespecificity, thus increasing the usefulness of this antibody if requiredat lower dilutions.

The CB2 and ECB antibodies reacted with the reciprocal immunizingpeptide. This was not unexpected since ECB was just an extension of CB2.Similarly, the CB2 and ECB antibodies were expected to react with theTCB peptide, the truncated version of CB2. However, the CB2 antibody didnot react with the TCB peptide and the ECB antibody had some reactivityto the TCB peptide that was beyond the lower limits of the assay. 70B′WP also did not react with the TCB peptide, but there was somereactivity with the CB2, ECB and CE peptides. This suggests that moreantigenic epitopes on the Hsp70B′ protein are responsible for thereactivity of 70B′ WP. The TCB antibody reacted with the immunizingpeptide and both the CB2 and ECB peptides.

TABLE 2 Relative Titre Index of Hsp70B′ Antibodies Rabbit AntibodyPurified Antibodies Serum Antibodies Hsp ProteinorHsp70B′ PeptideCB2Hsp70B′624-638

CEHsp70B′553-567 ECBHsp70B′618-638 TCBHsp70B′624-635 70B′ WPHsp70B′1-643SPA-812Hsp70A1-641

Hsp70B′ Protein ≧177

≧48 ≧41 ≧29 ≧182 ≧12

Hsc70 Protein 1

1 1 1 *1 *1

Hsp70A Protein 1 1 1 1 1 ≧2 ≧143

DnaK Protein 1 1 1 1 1 ≧2 *1

Grp78 Protein 1 1 1 1 1 1 1

Hsp71 Protein 1

1 1 1 *1 1

CB2 Peptide ≧92

1 ≧86 ≧37 ≧5 1 1 ECB Peptide ≧209

1 ≧129 ≧57 ≧9 1 1 TCB Peptide 1

1 *1 ≧243 1 1 1 CE Peptide 1

≧201 1 1 ≧7 1 1 *Expected to be >1 if actual titres were determined.

Irrelevant antibodies used to assess non-specific binding are shaded ingray. For purified antibodies, index values were calculated by dividingthe titre of the irrelevant antibody by the titre of the test antibody.For serum antibodies, the index values were calculated by dividing thetitre of the test antibody by the titre of the irrelevant antibody.

Determination of Antibody Specificity by Competition EIA

Selected Hsp70B′ antibodies were evaluated in a competition EIA asanother method for assessing antibody specificity. This competition EIAwas based on the 50% displacement method for calculating crossreactivity (Abraham, G.E., J. Clin. Endocrinol. Metab. 29:866-870,1969). As described by Abraham, a constant amount of antibody andlabeled standard (S) are incubated in the presence of varying doses ofunlabeled S or cross reactant (CR). The unlabeled S or CR is the“displacer” that competes with the labeled S for antibody binding sites.The bound labeled S is then quantified at each dose of unlabeled S orCR. Displacement curves are generated by plotting the % B/B₀ against thedose of unlabeled S or CR. B is the amount of bound labeled S in thepresence of added unlabeled S and B₀ is the amount of bound labeled S inthe absence of added unlabeled S (i.e. maximum bound labeled standard).In the 50% displacement method, cross reactivity is the ratio ofunlabeled S to CR doses that give 50% displacement of bound labeled S,expressed as a percentage.

A competition EIA was developed to determine the specificity of selectedHsp70B′ antibodies. Labeled standard is required for the competitionreaction described by Abraham. However, labeled Hsp70B′ protein wasunavailable for this study. Instead, the competition reaction wasmodified to measure the amount of antibody binding to a constant amountof S bound to a solid phase, in the presence of varying amounts of freeS or CR. Displacement curves were generated by plotting the % A/Amaxagainst the dose of the free displacer, where A was the amount ofantibody bound in the presence of displacer and Amax was the amount ofantibody bound in the absence of displacer. Similar to the Abrahammethod, cross reactivity was defined as the ratio of the free standardand cross reactant doses that resulted in 50% displacement (A/Amax) ofbound antibody.

Displacement curves for CB2 (FIG. 1A, purified rabbit antibody), CE(FIG. 1B) and 70B′ WP (FIG. 1 C) antibodies were generated with Hsp70B′protein (S) and five HSP70 homologs (CR): Hsp70A, Hsc70 , Grp78, DnaKand Hsp71. The concentration range of the free Hsp70B′ protein was 0.016-10 μg/ml whereas the range for the cross reactants was 0.8 -500 μg/ml.For all three Hsp70B′ antibodies, there was no distinct competition fromthe cross reactants when compared to Hsp70B′, despite using 50 fold morecross reactant at the highest dose. Under these assay conditions, a 50%displacement concentration could not be obtained for the cross reactantssince the average % A/Amax values throughout the cross reactantconcentrations ranged from 93-100%. It was unsuitable to redefine the50% displacement cutoff to 5% (i.e. % A/Amax of 95%) since variabilityof the assay could account for that level of displacement, rather thanbeing a true competition. In any case, higher cross reactantconcentrations beyond 500μg/ml are required for a more accurateassessment.

In Table 3, the percentage of cross reactivity with HSP70 homologs arepresented for the CB2, CE and 70B′ WP antibodies. Since the 50%displacement doses for the cross reactants were unobtainable, >500 μg/mlwas reported and 500 μg/ml was used for calculating the crossreactivity. The cross reactivity percentages are therefore actuallylower than the absolute reported values. Based on this data and underthese assay conditions, the CB2, CE and 70B′ WP antibodies were morereactive with Hsp70B′ by at least 758, 142 and 263 fold respectivelyover the tested HSP70 homologs. The CB2 antibody is notably morereactive with Hsp70B′ than the CE and 70B′ WP antibodies. This is likelybecause the CB2 antibody was affinity purified, whereas CE and 70B′ WPwere antisera. The CB2 antibody was competed with lower amounts of freestandard, suggesting that this preparation contained a greaterproportion of higher affinity antibodies as compared to the CE and 70B′WP antibodies.

The CB2 and CE antibodies were considered specific for Hsp70B′ whenexpressed in terms of a relative titre index (Table 2). Under thecompetition EIA conditions, these two antibodies can also be consideredspecific for Hsp70B′. The 70B′ WP antibody, however, was not specificfor Hsp70B′ at lower dilutions (e.g. 1:2000) by relative titre index.The competition assay used 70B′ WP at a dilution that was 16 foldhigher. This indicates that cross reactivity can essentially be “dilutedout” for this antibody and perhaps should be used at higher dilutions tomaximize specificity.

Displacement Curves for rabbit Hsp70B′ antibodies: CB2 (A), CE (B) and70B′ WP (C). HSP70 homologs were unable to significantly displace CB2,CE and 70B′ WP antibody binding to solid phase Hsp70B′. Under theseassay conditions, CB2, CE and 70B′ WP are specific for Hsp70B′.

TABLE 3 Specificity of Selected Hsp70B′ Antibodies in terms of CrossReactivity with Hsp70 Homologs Concentration of Rabbit Free DisplacerFree Displacer at Hsp70B′ (Standard or 50% A/Amax % Cross Antibody CrossReactant) (μg/ml) Reactivity CB2 Hsp70B′ (S) 0.66  100% (Purified) Hsp70(CR) >500 <0.13% Hsc70 (CR) >500 <0.13% Grp78 (CR) >500 <0.13% DnaK(CR) >500 <0.13% Hsp71 (CR) >500 <0.13% CE Hsp70B′ (S) 3.52  100%(Antisera) Hsp70 (CR) >500 <0.70% Hsc70 (CR) >500 <0.70% Grp78 (CR) >500<0.70% DnaK (CR) >500 <0.70% Hsp71 (CR) >500 <0.70% 70B′ WP Hsp70B′ (S)1.88  100% (Antisera) Hsp70 (CR) >500 <0.38% Hsc70 (CR) >500 <0.38%Grp78 (CR) >500 <0.38% DnaK (CR) >500 <0.38% Hsp71 (CR) >500 <0.38%

Cross reactivity was calculated by dividing the concentration of freestandard by the concentration of cross reactant at 50% A/Amax. The ratiowas then expressed as a percentage.

Determination of Antibody Specificity by Immunoblotting

Each of the Hsp70B′ antibodies detected their respective epitopes in thesynthetic peptide, the peptide-KLH conjugate, the recombinant Hsp70B′protein as well as in the native Hsp70B′ protein present in culturedcell lysates. Antibody binding to native Hsp70B′ protein in cell lysateswas determined by immunoblotting. Several control antibodies wereincluded in these experiments to validate the assays and inductionconditions. The reactivity profiles of the control antibodies issummarized in Tables 4 to 6. The anti-Hsc70 antibody (SPA-815; clone1B5) is a rat monoclonal antibody originally produced to the CHO(hamster) Hsc70 protein. This antibody was found to react only withHsc70 protein, no reactivity with any of the inducible isoforms, Grp78,DnaK, or Hsp71 was detected. Anti-Hsp70A (SPA-812) specific antibody isa rabbit polyclonal antibody produced to purified recombinant humanHsp70A protein. This antibody reacted only with the Hsp70A protein,there was no reactivity detected with Hsp70B′, Hsp70B fragment, Hsc70,Grp78, E. coli DnaK, and M. tuberculosis Hsp71 proteins in immunoblots.The third control antibody is a mouse monoclonal antibody (SPA-810;clone C92F3A-5) which was originally produced to human Hsc70 /Hsp70proteins purified from HeLa cells. This antibody was found to react withHsp70A and recombinant Hsp70B′, but not with Hsc70 , Hsp70B proteinfragment, Grp78, DnaK or Hsp71. Other control antibodies included arabbit Hsp110 polyclonal (SPA-1101), a mouse Grp75 monoclonal (SPA-825;clone 30A5), a mouse monoclonal specific for the ER retention signalpeptide KDEL (SPA-827; clone 10C3), a mouse DnaK monoclonal (SPA-880;clone XXX), and a mouse Hsp71 monoclonal (SPA-885; clone 5A8). TheHsp110 and Grp75 antibodies did not react with Hsp70A, Hsp70B′, Hsc70 ,Grp78, DnaK or Hsp71 proteins. The KDEL antibody reacted with Grp78protein, but not with Hsp70A, Hsp70B′, Hsc70 , DnaK or Hsp71 proteins.The DnaK and Hsp71 antibodies reacted respectively with DnaK and Hsp71proteins, but not with Hsp70A, Hsp70B′, Hsc70 , or Grp78 proteins. Usingthese control antibodies, Hsp110, Grp75, Hsc70 , and KDEL proteins weredetected in control and heat stressed human A431 cells, monkey Verocells, hamster CHO cells and bovine MDBK cells (Table 6). Hsp70A wasdetected in control A431, Vero and MDBK cells. It was not detected incontrol CHO cells. Hsp70A was detected at elevated levels in the heatstressed A431, Vero, CHO and MDBK cells.

TABLE 4 Summary of Anti-HSP70 Antibody Reactivity Profiles withDifferent HSP70 Family Members Hsc70 Hsp70A Hsp70B′ Hsp70B Grp78 DnaKHsp71 Reactivity Reactivity Reactivity Reactivity Reactivity ReactivityReactivity Epitope Rec. Rec. Rec. Rec. Rec. Rec. Rec. (Ab) Name Locationprotein^(∇) protein^(∇) protein^(∇) protein^(∇) protein^(∇) protein^(∇)protein^(∇) CB 624-638 0 0 3 0

0 ND ND CA 561-573 0 0 3 0 ND ND ND CD 561-576 2 1 3 0 ND ND ND CC546-559 0 0 3 3 ND ND ND NT  1-12 2 2 3 3 ND ND ND ^(♦)ECB 618-638

0

0 3 ND

0 ND ND ^(♦)TCB 624-635

0

0 2 ND

0 ND ND ^(♦)CE 553-567 0 0 3 ND 0 0 0 CB2 (rabbit) 624-638 0 0 3 ND 0 00 ^(♦)CB2 (goat) 624-638

0

0 2 ND

0 ND ND ^(♦)CB2 (mouse) 624-638

0

0

3 ND

0 ND ND *70B′ WP Hsp70B′ 0 0 2 ND 0 0 0  1-643 SPA-810 437-504 0 3 2 0 00 0 (Hsp70A) SPA-812 Multiple 0 3 0 0 0 0 0 Hsp70A SPA-815 Hsc70 3 0 0 00 0 0 SPA-1101 Hsp110 0 0 0 ND 0 0 0 626-640 SPA-825 Grp75 0 0 0 ND 0 00 SPA-827 KDEL 0 0 0 ND 1 0 0 SPA-880 DnaK 0 0 0 ND 0 2 0 SPA-885 Hsp710 0 0 ND 0 0 3 ^(∇)Relative reactivity as assessed by Western blottinganalysis of SDS-denatured recombinant proteins. Intensity of the antigenspecific bands was scored on a relative scale where 0 = no signaldetected and 3 = a very strong signal. ND = not determined.^(♦)Representative sera from one animal. *Pooled sera.

Determined with native protein in a cell lysate instead of recombinantprotein. Shaded Hsp70B′ antibodies are affinity purified.

The nine rabbit anti-peptide antibodies generated to Hsp70B′ epitopesreacted with Hsp70B′ recombinant protein in Western blot analysis (Table4). The antibodies also detected Hsp70B′ protein only in stressed celllysates prepared from human cell lines (Tables 5 and 6).

The immunoaffinity purified rabbit anti-CB and CB2 antibody preparations(epitope residues: 624-638) were found to be specific for the Hsp70B′protein; only a single 69 kDa protein is detected on Western blotsanalysis with complex protein mixtures from total human cell lysates.Although strong reactivity was seen with both native and recombinantHsp70B′, there was no reactivity with recombinant and/or native Hsc70 ,Hsp70A, DnaK, Hsp71, Hsp110, Grp75, Grp78 and other KDEL proteins(Tables 4 -6).

Representative CB2 sera samples from individual mice exhibitedspecificity to the native Hsp70B′ protein in a lysate (Table 5) whenassessed by immunoblotting. However, 3/10 CB2 immunized mice either didnot detect native Hsp70B′ or weakly reacted with other unknown proteinsin a lysate. The goat CB2 antisera also specifically detected nativeHsp70B′ protein, despite low anti-CB2 peptide titres. However, thereactivity of the goat antisera with native Hsp70B′ was lower, whencompared to the rabbit and mouse CB2 antibodies (Table 5).

In any case, CB/CB2 is a unique epitope that can be used reproducibly(i.e. separate occasions, different animals) to generate Hsp70B′specific antibodies. This epitope may be used to generate antibodies,such as monoclonals, of exquisite specificity and useful affinity. TheCB/CB2 epitope has already been used to produce mouse hybridomas thatreact with the CB/CB2 peptides. The reactivity of the hybridomas withHsp70A and Hsp70B′ proteins is currently being evaluated. Ideally, themonoclonal(s) will exhibit the same specificity as the polyclonal CBantibodies and will bind peptide, recombinant and native Hsp70B′ with aK_(a) in the range of 10⁴ -10¹²M⁻¹. The antibodies generated to thisepitope are excellent candidates for inclusion in the establishment ofrapid screening assays.

The peptide immunogens for the ECB and TCB antibodies were based on theCB/CB2 epitopes. The ECB peptide was an extended version of CB/CB2 bysix amino acids at the N-terminal end. When ECB sera from threeindividual rabbits were analyzed by immunoblots, all three rabbitsproduced antibody that reacted extremely well with purified recombinantHsp70B′ (Table 4) and native Hsp70B′ in a lysate (Table 5). However, ⅓antisera specifically detected Hsp70B′ in a lysate. The remaining 2antisera reacted very weakly with other unknown non-inducible proteinsin the cell lysates. This antibody (from 3/3 rabbits), however, did notreact with native Hsp70A, Hsc70 , Hsp110, Grp75, Grp78 or other KDELproteins. Any weak binding to the unknown lysate proteins can likely be“diluted out” or the antibody could be affinity purified to improvespecificity. If successful, the ECB antibody could be used inconjunction with other Hsp70B′ antibodies, such as CE, CA, or CC, forimmunoassay development. The ECB epitope would also be a good candidatefor a monoclonal antibody development.

Anti-TCB was generated with a CB/CB2 peptide that was truncated by threeamino acids at the C-terminal. Although the TCB antisera from 3/3rabbits reacted well to the immunizing peptide (Table 1), only 1/3rabbits produced antibody that reacted with recombinant (Table 4) andnative Hsp70B′ (Table 5), and an unknown non-inducible 40kDa protein inimmunoblots. The reactivity with the non-inducible protein can likely be“diluted out” or the antibody can be purified to improve specificity.The sera from the two other TCB immunized rabbits reacted with justrecombinant Hsp70B′ and not the native form (Table 5), suggestingdifferences in protein folding and surface exposure between therecombinant and native proteins. These two antisera also reacted withother unknown non-inducible proteins. The antisera from all threerabbits did not react with Hsp70A or Hsc70 . The lower success ratio(i.e. 1/3 rabbits detected native Hsp70B′) with the TCB peptide suggestsan importance for the three omitted GPI amino acids present on theCB/CB2 and ECB epitopes. Based on relative titre index, the CB2, 70B′ WPand ECB antibodies exhibited no to low reactivity with the TCB peptide(Table 2), but these three antibodies reacted with both recombinant andnative Hsp70B′. This supports the importance of the GPI residues forHsp70B′ antibody production, despite predictions of low antigenicity andsurface probability. Taken together, TCB polyclonals that recognizenative Hsp70B′ may be problematic to produce and resupply for industrialimmunoassay uses.

The anti-“CA” antibody (epitope residues: 561-573) recognizes the Hsp70Bprotein but not the Hsc70 , Hsp70A nor the Hsp70B (fragment) purifiedproteins. This antibody does, however, identify other unknown proteinsof lower and higher molecular weight. As long as the interpretation ofbinding reactivity of this antibody on immunoblots is restricted to awindow of 60 -80 kDa, this antibody will act as a sensitive and specificprobe in immunoblot analysis for the Hsp70B′ protein. None of the otherproteins seen on immunoblots with this antibody are stress inducibleunder the conditions of these experiments. The non-Hsp70B′ proteins aredetected equally well in both stressed and unstressed cell lysates.Although this antibody can be used in immunoblot analysis for thespecific detection of the Hsp70B′ protein the cross-reactivity of thisantibody for other cellular proteins decreases the usefulness of theantibody in immunoassay rapid screening tests. However, in combinationwith another specific antibody such as “CB”, this antibody may be auseful component in immunossays.

The anti-“CD” antibody (epitope residues: 561-576) recognizes bothrecombinant and native Hsp70B′ as well as the constitutive Hsc70protein. The inducible Hsp70A protein is also weakly recognized by thisantibody. In addition, this antibody will detect, on immunoblotanalysis, some unknown lower molecular weight constitutively expressedproteins. The large variation in “CA” and “CD” antibody is surprising asthe peptides used to generate these antibodies were very similar. The“CD” peptide is identical to the “CA” peptide with three exceptions; (a)the “CD” peptide lacks the additional N-terminal cysteine residue usedto facilitate coupling of the “CA” peptide to KLH, (b) the coupling toKLH was modified to coupling through the natural cysteine sequence atthe carboxy-terminus, and (c) three additional (DKC) residues were addedto the carboxyl-terminus of the “CD” epitope. These three additionalresidues are conserved in Hsc70 , Hsp70A as well as in Hsp70B′ sequencesand are predicted to lie within a hydrophobic region which is predictedin the Emini analyses to be antigenic. As the molecular weights of the 3proteins are different; 69, 72 and 73 for the Hsp70B′, Hsp70A and Hsc70proteins respectively, expression patterns of the different Hsp's may bedifferentiated with this antibody on immunoblot analysis. However, dueto the cross-reactivity of this antibody for other Hsp family members aswell as other cellular proteins this antibody would not be useful inrapid screening immunoassays.

The anti-“CC” antibody (epitope residues: 546-559) recognizes bothHsp70B (fragment) and Hsp70B′ recombinant proteins. This antibody doesnot recognize either the Hsc70 cognate protein nor the stress inducibleHsp70A protein. Although some additional unknown higher and lowermolecular weight proteins are detected on immunoblots with thisantibody, these proteins are also not inducible under these conditionsand do not fall within the 60-80 kDa window for evaluation. Thisantibody is, therefore, useful for immunoblot analysis but due tocross-reactivity with other cellular proteins the utility of thisantibody in immunoassay analysis is decreased. However, in combinationwith another specific antibody such as “CB”, this antibody may be auseful component in immunossays.

The CE antibody was made from a peptide which combined seven amino acidsfrom the C-terminal of CC and seven amino acids from the N-terminal ofCA, linked via a leucine residue. Specific Hsp70B′ binding was observedwith CE antisera from 1/3 rabbits in immunoblots (Table 5). There was noreactivity with Hsp70A, Hsc70 , Hsp110, Grp75, DnaK, Hsp71, Grp78 orother KDEL proteins. The CA and CC antibodies both detectednon-inducible proteins in addition to Hsp70B′; the CE antisera from thisparticular rabbit did not. Like the CB2 antibody, the CE antisera fromthis rabbit only detected human Hsp70B′ in lysates. There was noreactivity with any proteins in monkey, hamster or bovine cell lysates.CE antisera from another rabbit reacted with recombinant and to a lesserdegree with native Hsp70B′ (Table 5), but also weakly with othernon-inducible proteins. Interestingly, the more specific CE antisera hadan eight fold lower anti-CE peptide titre than the non specific CEantisera (Table 1). The CE antisera from the remaining rabbit reactedwith several non-inducible proteins in a lysate and very minimally withrecombinant Hsp70B′. Reactivity of this antisera with native protein wasinconclusive since many proteins were detected in the expected 70 kDamolecular weight range. Although the CE antibody has a lower successratio, a monoclonal antibody may be beneficial if it exhibits the samereactivity as the specific polyclonal. Because the locations of the CEand CB/CB2 or ECB peptides are distinct, specific monoclonal antibodiesto these epitopes would be choice components for two-site immunoassaydevelopment.

The “NT” antibody (epitope residues: 1-12) is an area of sequence ofrelative sequence homology within the Hsp70 family. This epitope has anoverall homology of less than 50% with other HSP70 family members,however, the areas of sequence identity are sequential. The anti-“NT”antibody is useful as an HSP70 family marker, but has no utility in thedifferential detection of different Hsp70 family members.

The 70B′ WP antibody specifically detected both recombinant and nativeHsp70B′ in immunoblots. When the antibody was used at higher dilutions(i.e., 1:20000), no cross reactivity with other HSP70 homologs or otherlysate proteins was observed. This antibody may be a useful component inimmunoassays if used at higher dilutions, purified or in conjunctionwith another specific antibody such as CB/CB2, ECB or CE. This antibodyhad relatively low titre index values with the CB, ECB, TCB and CEpeptides), but a high value with Hsp70B′ (Table 2). This suggests thatother specific epitopes, perhaps conformational, exist for Hsp70B′monoclonal antibody production that require whole protein immunizationsto exploit.

Summary of Hsp70B′Antibody Specificity. Table 7 summarizes theassessment of specificity for the rabbit Hsp70B′ antibodies with respectto the three methods performed: relative titre index, competition EIAand immunoblotting. Only one antibody, CB2 was considered specific forHsp70B′ in all three methods of assessment. Since CB was generated withthe same peptide sequence, it is likely that this antibody would also beconsidered Hsp70B′ specific for all three assessments methods. The ECB,CE and 70B′ WP antibodies could likely be purified or used at higherdilutions to maximize specificity. The CD and NT antibodies were notuseful for distinguishing Hsp70B′ from other HSP70 homologs,illustrating that the peptide approach is not 100% successful forgenerating Hsp70B′ antibodies.

TABLE 5 Reactivity of Hsp70B′, Hsp70A and Hsc70 Antibodies with 68 kDa(Hsp70B′) Protein from Unstressed and Heat Stressed Human Cell LysatesAntibody Antigen Animal Designation Epitope Number HeLa HeLa HS JurkatJurkat HS A431 A431 HS CB 624-638 N/A 0 +3  0 +3 0 +3 CA 561-573 N/A 0+3  0 +3 0 +3 CD 561-576 N/A 0 +3  0 +3 0 Not tested CC 546-559 N/A 0+3  0 +3 0 +3 NT  1-12 N/A +3  +3  +3  +3 Not tested Not tested ♦ECB618-638 1 0 3 Not tested Not tested Not tested Not tested 2 0 3 Nottested Not tested Not tested Not tested 3 0 3 Not tested Not tested Nottested Not tested ♦TCB 624-635 1 0 2 Not tested Not tested Not testedNot tested 2 0 0 Not tested Not tested Not tested Not tested 3 0 0 Nottested Not tested Not tested Not tested ♦CE 553-567 1 0 3 Not tested Nottested 0  3 2 0 1 Not tested Not tested Not tested Not tested 3 Nottested Not tested Not tested Not tested 2  2 CB2 (rabbit) 624-638 N/ANot tested Not tested 0  3 0  3 ♦CB2 (goat) 624-638 1 0 1 Not tested Nottested Not tested Not tested

CB2 (mouse) 624-638 1 0 3 Not tested Not tested Not tested Not tested 20 3 Not tested Not tested Not tested Not tested 3 0 2 Not tested Nottested Not tested Not tested 4 0 1 Not tested Not tested Not tested Nottested 5 0 0 Not tested Not tested Not tested Not tested *70B′ WPHsp70B′ N/A Not tested Not tested Not tested Not tested 0  2  1-643SPA-810 Hsp70A N/A +2  +3  +2  +3 +2  +3 437-504 SPA-812 Hsp70A N/A +3 +3  +1  +2 +2  +2 Multiple SPA-815 Hsc70 N/A +3  +3  +2  +3 +3  +3Unknown In Table 5, reactivity levels were assessed by immunoblottinganalysis of 10-20 μg lysate. Intensity of antigen specific bands wasscored on a relative scale where 0 = no signal and 3 = a very strongsignal. ♦Relative reactivity levels are reported for unpooled serumantibodies from individual animals.

Relative reactivity levels are reported for 5/10 mice. *Relativereactivity levels are reported for pooled serum antibodies. Shaded areasrepresent inconclusive binding to Hsp70B′. N/A in the Animal Numbercolumn indicates purified antibody.

TABLE 6 Reactivity of Hsp Antibodies with Control and Heat StressedMammalian Cell Lysates Antibody Location A431 A431 HS Vero Vero HS CHOCHO HS MDBK MDBK HS CB2 Hsp70B′ 0 +3 0 0 0 0 0 0 624-638 CE Hsp70B′ 0 +30 0 0 0 0 0 553-567 70B′ WP Hsp70B′ 0 +2 0 0 0 0 0 0  1-643 SPA-1101Hsp110 +2 +2 +2 +1 +2 +2 +2 +2 626-640 SPA-810 Hsp70A +2 +3 +2 +3 0 +2+1 +3 437-504 SPA-812 Hsp70A +2 +3 +2 +3 0 +1 +1 +2 Multiple SPA-815Hsc70 +3 +3 +3 +3 +3 +3 +3 +3 Unknown SPA-825 Grp75 +3 +3 +3 +3 +3 +3 +3+3 Unknown SPA-827 KDEL +1 +2 +2 +2 +3 +3 +1 +1 649-654 SPA-880 DnaK 0 00 0 0 0 0 0 Unknown SPA-885 Hsp71 0 0 0 0 0 0 0 0 Unknown Reactivitylevels were assessed by immunoblotting analysis of 10-20 mg lysate.Intensity of antigen specific bands was scored on a relative scale where0 = no signal and 3 = a very strong signal. {umlaut over ( )} Relativereactivity levels are reported for unpooled serum antibodies fromindividual animals. § Relative reactivity levels are reported for 5/10mice. * Relative reactivity levels are reported for pooled serumantibodies. Shaded areas represent inconclusive binding to Hsp70B′. N/Ain the Animal Number column indicates purified antibody.

TABLE 7 Summary of Rabbit Hsp70B′ Antibody Specificity Method forSpecificity Assessment Antibody Location Relative Competition NameHsp70B′ Titre Index EIA Immunoblot CB 624-638 ND ND specific forrecombinant and native Hsp70B′ CA 561-573 ND ND detects other proteinsin cell lysates; i.e. not specific CD 561-576 ND ND cross reactivitywith other HSP70 family members and cellular proteins; i.e. not specificCC 546-559 ND ND detects other proteins in cell lysates; i.e. notspecific NT  1-12 ND ND detects other HSP70 family members; i.e. notspecific ECB 618-638 Specific ND One bleed for specific for Hsp70B′Hsp70B′; other bleeds weakly detect other proteins, but can likely bediluted out TCB 624-635 Specific ND Not specific; for 2/3 bleeds didHsp70B′ not detect native Hsp70B′ CE 553-567 Specific Specific for Onebleed for Hsp70B′ specific for Hsp70B′ Hsp70B′; 2/3 bleeds not specificCB2 624-638 Specific Specific for Specific for for Hsp70B′ Hsp70B′Hsp70B′ 70B′  1-643 Not Specific for Specific for WP specific Hsp70B′Hsp70B′ for when used at when used at Hsp70B′ a high dilution a highdilution at lower dilutions

Relative reactivity levels were assessed by Western blotting analysis of10 μg of total cell lysate. Immunoblots were probed with the Hsp70B′antibodies, CB2, CE and 70B′ WP, as well as anti-Hsp antibodies specificfor Hsp110, Hsp70 A, Hsc70 , Grp75, KDEL, DnaK and Hsp71. The CEantibody used for this study was the sera specific for Hsp70B′ from onerabbit. Immunoblots were developed by ECL. Intensity of the antigenspecific bands was scored on a relative scale where 0=no signal detectedand 3=a very strong signal.

Hsp70B′ Protein: Distribution and Induction Conditions. As can be seenin Tables 5, 6 and 8, the Hsp70B′ antibodies (NT, CA, CB, CC, CD, ECB,TCB, CB2, CE and 70B′ WP) detect the Hsp70B′ protein only in stressedhuman tissues, no reactivity is detected in non-stressed cells ortissues.

TABLE 8 Reactivity of Anti-Hsp70B′ “CB” Antibody with a Panel ofDifferent Cell Lysates from a Variety of Different Tissue Sources andDifferent Species Reactivity Cell Line (non- Reactivity DesignationDescription Species stressed) (stressed) HeLa Epitheloid carcinoma,Human Negative +3 cervix A431 Epidermoid carcinoma Human Negative +3Jurkat Adult T-cell leukemia Human Negative +2 H4 Neuroglioma, brainHuman Negative +1 MCF7 Breast adenocarcinoma, Human Negative +(weak)pleural effusion HMEC Mammary epithelial Human Negative Not tested cells(normal) HRCE Renal cortical Human Negative Not tested epithelial cells(normal) RPTEC Renal proximal tubule Human Negative Not testedepithelial cells (normal) NHBE Bronchial epithelial Human Negative Nottested cells (normal) PrEC Prostate epithelial Human Negative Not testedcells (normal) NHEK-Ad Epidermal Human Negative Not tested keratinocytesadult (normal) NHEK-Neo Epidermal Human Negative Not testedkeratinocytes neo (normal) NHEF-Neo Epidermal Human Negative Not testedpool keratinocytes neo pool (normal) NHDF-Ad Dermal fibroblast HumanNegative Not tested adult (normal) NHDF-Neo Dermal fibroblast HumanNegative Not tested neo (normal) HMVEC-d Microvascular Human NegativeNot tested Ad endothelial adult (normal) HMVEC-d Microvascular HumanNegative Not tested Neo endothelial neo (normal) NHEM Melanocytes NeoHuman Negative Not tested (normal) HPAEC Pulmonary artery Human NegativeNot tested endothelial cells (normal) HCAEC Coronary artery HumanNegative Not tested endothelial cells (normal) HIAEC Lliac artery HumanNegative Not tested endothelial cells (normal) HAEC Aortic endothelialHuman Negative Not tested cells (normal) HMVEC-L Lung microvascularHuman Negative Not tested endothelial cells (normal) HUVEC Umbilicalvein Human Negative Not tested endothelial cells (normal) HUAECUmbilical artery Human Negative Not tested endothelial cells (normal)AoSMC Aortic artery Human Negative Not tested smooth muscle cells(normal) BSMC Bronchial/trachial Human Negative Not tested smooth musclecells (normal) CASMC Coronary artery Human Negative Not tested smoothmuscle cells (normal) PASMC Pulmonary artery Human Negative Not testedsmooth muscle cells (normal) UASMC Umbilical artery Human Negative Nottested smooth muscle cell (normal) UtSMC Uterine smooth muscle HumanNegative Not tested cells (normal) SkMC Skeletal muscle cells HumanNegative Not tested (normal) Liver (normal tissue) Human Negative Nottested Lung (normal tissue) Human Negative Not tested Brain (normaltissue) Human Negative Not tested Kidney (normal tissue) Human NegativeNot tested Testis (normal tissue) Human Negative Not tested Ovary(normal tissue) Human Negative Not tested Heart (normal tissue) HumanNegative Not tested Spleen (normal tissue) Human Negative Not testedVERO Kidney Monkey Negative Negative L929 Connective tissue MouseNegative Negative 3T3 Embryo Mouse Negative Negative Rat2 Embryo RatNegative Negative GPC-16 Colon, adenocarcinoma Guinea Negative Negativepig MDOK Kidney Sheep Negative Negative MDBK Kidney Cow NegativeNegative ESK4 Kidney Pig Negative Negative

As can be seen in Tables 3 and 4 the Hsp70B′ antibodies (NT, CA, CB, CCand CD) detect the Hsp70B′ protein only in stressed human tissues, noreactivity is detected in non-stressed cells or tissues. Eight differentnormal human tissues, 27 samples of cell line lysates derived fromnormal human sources, five samples of cell line lysates derived fromneoplastic tissues and seven other mammalian species were evaluated forthe expression of the Hsp70B′ protein. The Hsp70B′ protein was notdetected by the anti-Hsp70B′ antibody “CB” in the 48 samples obtainedfrom “normal” or “unstressed” cells. If these cells are exposed toelevated temperatures, the Hsp70B′ protein is expressed in all of theheat-shocked human cell lines evaluated. These results are consistentwith earlier findings at the mRNA level, using specific oligonucleotideprobes (Leung et al., Biochem. J. 267: 125-132, 1990; Leung et al.,Genomics 12:74-79, 1992). These investigations did not detect thepresence of any hsp70B′ mRNA in the unstressed cells.

The temperature threshold of Hsp70B′ protein expression in heat treatedHeLa cells following recovery for 0, 2.5, 5, 16 and 24 hours at 37° C.was investigated. In this study the level of expression of the Hsp70B′protein was greatest at the 16 hour (+2) timepoint. Therefore if asignificant level of Hsp70B′ protein was detected, this would imply thatthe cells or individual tested was either currently or had recently beenexposed to a significant stressor and not due to a stressful situationoccurring far in the past. The stress response does, however, persistlong enough to be potentially useful as a diagnostic probe. Thepersistence of the Hsp70B′ protein in cells following stress is similarto the results described for Hsp70A protein in recovering peripheralblood lymphocytes (Bratton et al., Int. J. Hyperthermia 13(2): 157-168,1997). These investigators found that the Hsp70A protein persisted forat least 48 hours and reached maximal expression at 12 hours.

TABLE 9 Investigation of the Induction^(∇) of Hsc70, Hsp70A and Hsp70B′Proteins* in Human Cell Lines Under Different Induction Conditions CellLine HeLa Cells Jurkat Cells A431 Cells Antigen Hsc70 Hsp70A/B′ Hsp70AHsp70B′ Hsc70 Hsp70A/B′ Hsp70A Hsp70B′ Hsc70 Hsp70A/B′ Hsp70A Hsp70B′Non- +3 +2 +2 0 +3 +1 +1 0 +3 +2 +2 0 stressed Heat +3 +2 +2 +1 +3 +2 +2+3 +3 +3 +2 +3 (430 C.) 20 min. Heat +3 +3 +3 +3 +3 +2 +2 +3 +3 +3 +2 +3(430 C.) 120 min. Azetidine +3 +3 +3 +1 +3 +2 +2 +1 +3 +3 +2 +2 (5 μM, 5hours) CdCl2 +3 +3 +3 +2 +3 +2 +2 +3 +3 +3 +2 +1 (100 μM, 2 hours) ZnCl2+3 +3 +3 +1 +3 +1 +2 +1 +3 +3 +2 +2 (250 μM, 2 hours) ^(∇)Relativeprotein levels as assessed by Western blotting analysis of 20 ug oftotal cell lysate. Intensity of the antigen specific bands was scored ona relative scale where 0 = no signal detected and 3 = a very strongsignal

TABLE 10 Dose Response Curve Showing the Relative Induction of HumanHsp70 Family Members in HeLa Cells Heat Stressed at DifferentTemperatures. Heatshock Hsp70A/ Hsp70B′/ Hsp70B′/ Hsp70B′/ temperatureHsc70 Hsp70B′ Hsp70A Hsp70B Hsp70B Hsp70B (° C.) SPA-815 SPA-810 SPA-812CB CA CC 37 3 3 2 0 0 0 38.5 3 3 2 0 0 0 40 3 3 2 0 0 0 41.5 3 3 2 2 1 243 3 3 2 3 3 3 44.5 3 3 2 3 3 3 * Relative protein levels as assessed byWestern blotting analysis of 20 ug of total cell lysate. Intensity ofthe antigen specific bands was scored on a relative scale where 0 = nosignal detected and 3 = a very strong signal)

The relative induction of the Hsc70 , Hsp70A and Hsp70B′ proteins underdifferent conditions were evaluated in HeLa, Jurkat and A-431 human celllines (Tables 9 and 10). It can be seen that the induction of theHsp70B′ protein was different than that seen for other HSP70 isoforms.Hsp70B′ protein was not present in unstressed cells and was induced onlyin response to cellular stress. The expression of the constitutive Hsc70protein is not affected by exposure to the proline analogue azetidine,to the heavy metals CdCl₂ or ZnCl₂ or to increased temperature. Theinducible Hsp70A stress protein appears to be expressed at high levelsbasal levels in unstressed cells as has been found previously by otherinvestigators (Turman et al., Biochemical and Molecular Medicine60:49-58, 1997). Changes to the level of Hsp70A protein expression wasfound to be obscured in these investigations by the high basal level ofexpression of this protein. The induction as a result of stresstreatment is consistent with published findings. The extent to whichHsp70A expression is induced by heat shock is inversely correlated withinitial levels of Hsp70A (Turman et al., Biochemical and MolecularMedicine 60:49-58, 1997). Therefore, in human cells, high basalexpression of Hsp70A may prevent further induction of HSP70 after heatshock.

The Hsp70B′ protein was not present in unstressed cells. Slighttemperature increases up to 40° C. did not elicit a response. Once thethreshold of 41.5° C. was reached, all three human cell lines (Hela,Jurkat and A-431) responded by expressing the Hsp70B′ protein. Thisinduction threshold of Hsp70B′ expression is different than thethreshold described previously in which hsp70B′ mRNA levels weremeasured using specific oligonucleotides (Leung et al., Biochem. J.267:125-132, 1990). These investigators reported that hsp70B′ mRNA wasstrongly induced at 45° C. and was not detectable after 42° C.treatment. Furthermore, they found only trace amounts of hsp70B′ mRNAafter CdCl₂ treatment in contrast to the protein data reported here(Table 5). The Hsp70B′ protein was shown to be induced by the prolineanalog azetidine as well as the heavy metals CdCl₂ and ZnCl₂, all knowninducers of stress proteins. This discrepancy in the induction thresholdas determined at the genomic and protein levels is likely due to theinherent technical differences in the two techniques. Previous studieslooking at regulation of the hsp70A and hsc70 genes have not yielded aconsistent set of results (Hansen et al., Exp. Cell Research192:587-596, 1991; Mangurten et al., Cell Stress & Chaperones2(3):168-174, 1997). Depending on the particular system and at whatlevel expression was examined, hsc70 expression can either increase ordecrease following treatment with agents to induce differentiation. Instudies of the differential expression of Hsp70A in wound healing, itwas found that whilst hsp70A mRNA did not show significant correlationwith healing, a strong correlation was seen between well healing woundsand expression of the Hsp70A protein (Oberringer et al., Biochemical andBiophysical Research Communications 214(3):1009-1014, 1995). It would bepreferable to measure the protein, since the protein response persistswhereas the mRNA has been reported to have a very short half-life(Bratton et al., Int. J. Hyperthermia 13(2):157-168, 1997).

At the level of transcription the hsp70A gene is regulated throughtranscription factors other than HSF's under non-stressed conditions andtherefore, the Hsp70A protein is detected even in the physiologicalstate (Hansen et al., Exp. Cell Research 192:587-596, 1991). The hsp70B′gene is regulated exclusively by the association of HSF's and the heatshock element, allowing no constitutive expression (Suzuki et al.,Radiation Research 149:195-201, 1998). The promoter regions of hsp70B′and hsp70B genes differ extensively from the hsp70A gene in that theylack TATA and CAAT boxes which are believed to contribute to the basalexpression of Hsp70A (Wu et al., Proc. Natl. Acad. Sci., USA83(3):629-633, 1986; Greene et al., Mol. Cell Biol. 7(10):3646-55,1987). The two hsp70B DNA homologs display differences in their 5′regions as well as several changes within key promoter sequences. Thehsp70B′ gene has been shown to have a 19-nucleotide-residue insertion inthe hsp70B gene that lies within the heat-shock element of the hsp70BDNA sequence (Leung et al., Genomics 12: 74-79, 1992). Elevated mRNAlevels do not always translate into increased protein levels due toregulation at the transcriptional and/or post-transcriptional levels(Oberringer et al., Biochemical and Biophysical Research Communications214(3):1009-1014, 1995). Using the anti-Hsp70B′ antibodies inimmunochemical testing procedures, therefore, allows a more sensitive,longer lasting quantitative evaluation of Hsp70B′ protein levels thanthat found using specific oligonucleotides to evaluate mRNA levels.

The Hsp70B′ protein has been shown to be expressed only followingsignificant stress on the cell or organism, whether this stress iscaused by elevated heat or exposure to heavy metals or toxic chemicals.The Hsp70B′ antibodies of the present invention offer a uniqueopportunity to use these naturally occurring biomarkers to evaluate thestress on a system. The nature of the stress need not be known.Monitoring the Hsp70B′ biomarker provides a prognostic indicator of thegeneral “wellness” of the cell or organism and indicate when asignificant perturbation has occurred. As biomarkers, Hsp's providessensitive early-warning of toxicity, perhaps allowing intervention at anearlier more tractable stage of the problem.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually incorporated by reference. From the foregoing, it will beevident that, although specific embodiments of the invention have beendescribed herein for purposes of illustration, various modifications maybe made without deviating from the spirit and scope of the invention.

1. An isolated peptide consisting of five or more consecutive amino acidresidues within one of the following amino acid sequences:VPGGSSCGTQARQGDPSTGPI; (SEQ ID NO:1) RDKIPEEDRRKMQDKC; (SEQ ID NO:4)AHVFHVKGSLQEESLRDKIPEEDRRKMQ; (SEQ ID NO:6) or wherein said peptide isan epitope from Hsp 70B′ protein MQAPRELAVGID. (SEQ ID NO:8)